Aminotriazolopyridine for use in the treatment of inflammation, and pharmaceutical compositions thereof

ABSTRACT

The present invention relates to the compound according to Formula I, and to its use in medicine, in particular in the treatment of inflammatory conditions, autoimmune diseases, proliferative diseases, allergy, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. In particular, the compound inhibits JAK a family of tyrosine kinases, and more particularly JAK1. The present invention also provides pharmaceutical compositions comprising the compound, methods for the prophylaxis and/or treatment of diseases involving inflammatory conditions, autoimmune diseases, proliferative diseases, allergy, transplant rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering the compound.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the priority of co-pending provisionalapplication U.S. Ser. No. 61/663,520 filed on Jun. 22, 2012, and thedisclosure of said application is incorporated by reference herein inits entirety. Applicants claim the benefits of said application under 35U.S.C. §119(e).

FIELD OF THE INVENTION

The present invention relates to the medical use of a compound of theinvention according to Formula I. In particular the present inventionrelates to the use of a compound of the invention according to Formula Ifor the treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons. In particular, the compound inhibits JAK, a family oftyrosine kinases, and more particularly JAK1. The present invention alsoprovides pharmaceutical compositions comprising the compound and methodsfor the prophylaxis and/or treatment of diseases including inflammatoryconditions, autoimmune diseases, proliferative diseases, allergy,transplant rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 or interferons by administering a compound ofthe invention according to Formula I.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transducecytokine signaling from membrane receptors to STAT transcriptionfactors. Four JAK family members are described, JAK1, JAK2, JAK3 andTYK2. Upon binding of the cytokine to its receptor, JAK family membersauto- and/or transphosphorylate each other, followed by phosphorylationof STATs that then migrate to the nucleus to modulate transcription.JAK-STAT intracellular signal transduction serves the interferons, mostinterleukins, as well as a variety of cytokines and endocrine factorssuch as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. etal. (2008)).

The combination of genetic models and small molecule JAK inhibitorresearch revealed the therapeutic potential of several JAKs.

JAK1 is a target in the immuno-inflammatory disease area. JAK1heterodimerizes with the other JAKs to transduce cytokine-drivenpro-inflammatory signaling. Therefore, inhibition of JAK1 is of interestfor immuno-inflammatory diseases with pathology-associated cytokinesthat use JAK1 signaling, such as IL-6, IL-4, IL-5, IL-12, IL-13, IL-23,or IFNgamma, as well as for other diseases driven by JAK-mediated signaltransduction.

In the JAK family members' roles, some overlap exists, since mostsignaling pathways involve more than one JAK, however for some growthfactors such as erythropoietin and thrombopoietin, only JAK2 isinvolved.

JAK3 plays a major role in blocking immune function via transmission ofsignals generated by interleukin (IL)-2.

On the other hand, TYK2 would appear to work in combination with JAK2and JAK3 in order to transduce signaling of cytokines such as IL-12 andIL-23.

The role of JAK enzymes has been mostly studied using mice where each ofthe JAK family members has been deleted. JAK1 knockout mice exhibit aperinatal lethal phenotype and also have defective lymphoid developmentand function as a result of defective signaling by cytokines throughJAK1. JAK2 deficiency results in embryonic lethality at day 12 as aresult of a failure in definitive erythropoiesis. JAK3-deficient micehave severe combined immunodeficiency (SCID) phenotype but do not havenon-immune defects. (Verstovsek, 2009, Hematology Am Soc Hematol EducProgram., 636-42).

As has been observed with pan JAK inhibitors, non selective inhibitionmay be linked to side effects such as anemia, an increased rate ofinfections, lower neutrophil and lymphocyte counts, a decrease inhaemoglobin, and elevated cholesterol levels, (Elie Dolgin, 2011, NatureReviews Drug Discovery 10, 717-718).

Therefore, the development of a selective JAK inhibitor would bebeneficial in order to minimize such side effects.

BACKGROUND OF THE INVENTION

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of a RA therapy, therefore, is not only to slow down the disease butto attain remission in order to stop the joint destruction. Besides theseverity of the disease outcome, the high prevalence of RA (˜0.8% ofadults are affected worldwide) means a high socio-economic impact. (Forreviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt(2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).

JAK1 is implicated in intracellular signal transduction for manycytokines and hormones. Pathologies associated with any of thesecytokines and hormones can be ameliorated by JAK1 inhibitors. Hence,several allergy, inflammation and autoimmune disorders might benefitfrom treatment with compounds described in this invention includingrheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathicarthritis, osteoarthritis, asthma, chronic obstructive pulmonary disease(COPD), tissue fibrosis, eosinophilic inflammation, esophagitis,inflammatory bowel diseases (e.g. Crohn's disease, ulcerative colitis),transplant, graft-versus-host disease, psoriasis, myositis, psoriaticarthritis, ankylosing spondylitis, juvenile idiopathic arthritis, andmultiple sclerosis (Kopf et al., 2010).

Psoriasis is a disease that can affect the skin. The cause of psoriasisis not fully understood, however, it is believed that it is an immunemediated related disease linked to the release of cytokines, inparticular TNFα, which causes inflammation and rapid reproduction of theskin cells. This hypothesis has been corroborated by the observationthat immunosuppressant medication can clear psoriasis plaques (Zenz R,Eferl R, Kenner L, et al. (2005). “Psoriasis-like skin disease andarthritis caused by inducible epidermal deletion of Jun proteins”.Nature 437 (7057): 369-75)

Psoriasis can also cause inflammation of the joints, which is known aspsoriatic arthritis. Between 10-30% of all people with psoriasis alsohave psoriatic arthritis (Committee for Medicinal Products for Human Use(CHMP) (18 Nov. 2004). “Guideline on Clinical Investigation of MedicinalProducts indicated for the treatment of Psoriasis”). Because of itschronic recurrent nature, psoriasis is a challenge to treat. It hasrecently been demonstrated that inhibition of JAK could result insuccessful improvement of the psoriatic condition. (Punwani et al.,(2012) “Preliminary clinical activity of a topical JAK1/2 inhibitor inthe treatment of psoriasis” J Am Acad Dermatol., 67, 4, 658-664).

Inflammatory bowel disease (IBD) is a group of inflammatory conditionsof the colon and small intestine. The major types of IBD are Crohn'sdisease and ulcerative colitis. Recently, it has been found viagenome-wide association (GWAS) studies that T cell protein tyrosinephosphatase (TCPTP) is a JAK/STAT and growth factor receptor phosphatasethat has been linked to the pathogenesis of type 1 diabetes, rheumatoidarthritis, and Crohn's disease by GWAS (Zikherman et al., J Clin Invest.2011 December; 121(12):4618-21). Therefore, inhibition of the JAKpathway might provide a way of treating IBD.

JAK family members have been implicated in additional conditionsincluding myeloproliferative disorders (O'Sullivan et al, 2007, MolImmunol. 44(10):2497-506), where mutations in JAK2 have been identified.This indicates that inhibitors of JAK in particular JAK2 may also be ofuse in the treatment of myeloproliferative disorders. Additionally, theJAK family, in particular JAK1, JAK2 and JAK3, has been linked tocancers, in particular leukaemias e.g. acute myeloid leukaemia(O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506; Xiang et al.,2008, “Identification of somatic JAK1 mutations in patients with acutemyeloid leukemia” Blood First Edition Paper, prepublished online Dec.26, 2007; DOI 10.1182/blood-2007-05-090308) and acute lymphoblasticleukaemia (Mullighan et al, 2009)), cutaneous T-cell lymphoma (Zhang etal., 1996, PNAS, 93, 9148-9153) or solid tumours e.g. uterineleiomyosarcoma (Constantinescu et al., 2007, Trends in BiochemicalSciences 33(3): 122-131), prostate cancer (Tam et al., 2007, BritishJournal of Cancer, 97, 378-383) and breast cancer (Berishaj et al.,2007, Breast Cancer Research 9: R32). These results indicate thatinhibitors of JAK, in particular of JAK1, may also have utility in thetreatment of cancers (leukaemias and solid tumours e.g. uterineleiomyosarcoma, prostate cancer).

In addition, Castleman's disease, multiple myeloma, mesangialproliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma arelikely due to hypersecretion of the cytokine IL-6, whose biologicaleffects are mediated by intracellular JAK-STAT signaling (Tetsuji Naka,Norihiro Nishimoto and Tadamitsu Kishimoto, Arthritis Res 2002, 4 (suppl3):S233-S242). This result shows that inhibitors of JAK, may also findutility in the treatment of said diseases.

The current therapies are not satisfactory and therefore there remains aneed to identify further compounds that may be of use in the treatmentof inflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, in particularrheumatoid arthritis.

Additionally, these conditions are chronic conditions which require longterm therapy, and repeated intake of the drug. Long term treatment mightbe a heavy burden on the patient and the practitioner alike, since thepatient might be or become intolerant to the drug, and furthermore highdosage, or high dosage frequency may result in uncomfortable sideeffects, and/or low patient compliance, where the patient mayoccasionally, deliberately or accidentally, miss a dose. The impact ofnon-adherence varies across chronic illnesses, and ranges from minimalto very significant. (Ingersoll et al., 2008 J Behav Med.; 31(3):213-224).

Therefore, there is a need to identify more compounds to reinforce thearsenal of the practitioner, and compounds with low frequency dosageregimen to improve the life of the patients.

In the quest to discover new medicines, criteria are often set toidentify the best suitable candidate, thus many compounds are rapidlyassessed in an in vitro model, and equally rapidly discarded if they donot meet said criteria. In vitro studies usually have a higherthroughput than in vivo studies and greatly help with the decisionmaking process. Thus the in vitro model is usually expected to bepredictive of the in vivo behavior of the drug, and compounds whichprima facie would not appear to offer a suitable profile in vitro arediscarded. In this context, the compound according to Formula I wheninvestigated showed in vitro profile of low interest, however, in vivostudies revealed unexpected properties in humans specifically.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the compound of theinvention according to Formula I may be useful as a medicament. In aparticular aspect, the compound of the invention according to Formula Iis an inhibitor of JAK, and more particularly JAK1.

The present invention also provides methods for the production of thecompound of the invention according to Formula I, pharmaceuticalcompositions comprising the compound of the invention according toFormula I and methods for the prophylaxis and/or treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, in particularrheumatoid arthritis, by administering the compound of the inventionaccording to Formula I.

Accordingly, in a first aspect of the invention, a compound of theinvention is provided for use in medicine having a Formula I:

In another embodiment, the compound of the invention may be prepared ina therapeutic combination, with a further therapeutically activeingredient. The further therapeutically active ingredient may be acompound for the treatment of the same conditions and diseases outlinedherein with respect to the compound of formula I. In a particularembodiment, the further therapeutically active ingredient may be acompound for the treatment of arthritis. In a most particular aspect,the further therapeutically active ingredient is a compound for thetreatment of rheumatoid arthritis.

The compound of the invention according to Formula I, surprisingly,exhibits in vivo in human a very different profile from other animalspecies, which contrasts with in vitro predictions. Indeed, in vivo ithas been demonstrated that in human, the apparent terminal half life ofthe compound of the invention according to Formula I is significantlylonger than in the other animal species by at least 3 fold. This causesaccumulation in human resulting in a maintained therapeutic effect overan extended period of time, thereby allowing once daily to once weeklydosing. Thus the compound of the invention according to Formula I mayprovide advantages including a low frequency dosage regimen and/orincreased patient compliance. In particular, the impact of nonadherence, if the patient misses a dose, might be reduced.

In a particular aspect, the compound of the invention according toFormula I is provided that may be used in a method for the prophylaxisand/or treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons, in particular rheumatoid arthritis.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising the compound of the invention according toFormula I, and a pharmaceutical carrier, excipient or diluent. In aparticular aspect, the pharmaceutical composition may additionallycomprise further therapeutically active ingredients suitable for use incombination with the compound of the invention according to Formula I.In a more particular aspect, the further therapeutically activeingredient is a compound for the treatment of arthritis. In a mostparticular aspect, the further therapeutically active ingredient is acompound for the treatment of rheumatoid arthritis.

Moreover, the compound of the invention according to Formula I, usefulin the pharmaceutical compositions and treatment methods disclosedherein, is pharmaceutically acceptable as prepared and used.

In a further aspect of the invention, this invention provides a methodof treating a mammal, in particular humans, susceptible to or afflictedwith a condition selected from among those listed herein, andparticularly inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, moreparticularly rheumatoid arthritis, which method comprises administeringa therapeutically effective amount of the pharmaceutical composition orcompound of the invention according to Formula I as described herein.

The present invention also provides pharmaceutical compositionscomprising the compound of the invention according to Formula I, and asuitable pharmaceutical carrier, excipient or diluent. In a particularaspect, the pharmaceutical composition is for use in the prophylaxisand/or treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons, in particular rheumatoid arthritis.

In additional aspects, this invention provides methods for synthesizingthe compound of the invention of according to Formula I, withrepresentative synthetic protocols and pathways disclosed later onherein.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description,including the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the rat CIA Clinical Score after treatment with thecompounds disclosed herein.

FIG. 2: Shows the evolution of ankle diameter in the rat CIA model aftertreatment with the compounds disclosed herein.

FIG. 3: Shows the in vitro metabolic profile of the compound accordingto Formula II

FIG. 4: Shows the ratio of exposure (expressed as AUC) for FormulaI:Formula II measured on administration of the compound according toFormula II.

FIG. 5: Shows the values for the exposure levels of the disclosedcompounds after administration of the compound according to Formula IIexpressed as a multiple of the IC₅₀ value.

FIG. 6: Shows the combined values for the exposure levels over a 24 hperiod of the compounds according to Formula I and Formula II afteradministration of the compound according to Formula II expressed as amultiple of the IC₅₀ value.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions arc intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the term ‘substituted’ is to be defined as set out below. It should befurther understood that the terms ‘groups’ and ‘radicals’ can beconsidered interchangeable when used herein.

The articles ‘a’ and ‘an’ may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein the term ‘JAK’ relates to the family of Janus kinases(JAKs) which are cytoplasmic tyrosine kinases that transduce cytokinesignaling from membrane receptors to STAT transcription factors. FourJAK family members are described, JAK1, JAK2, JAK3 and TYK2 and the termJAK may refer to all the JAK family members collectively or one or moreof the JAK family members as the context indicates.

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable salt’ refers to a salt of the compoundaccording to Formula I that is pharmaceutically acceptable and thatpossesses the desired pharmacological activity of the parent compound.In particular, such salts are non-toxic may be inorganic or organic acidaddition salts and base addition salts. Specifically, such saltsinclude: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term‘pharmaceutically acceptable cation’ refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which the compound according to Formula I isadministered.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding. Conventional solvents include water, ethanol,acetic acid and the like. The compound according to Formula I may beprepared e.g. in crystalline form and may be solvated or hydrated.Suitable solvates include pharmaceutically acceptable solvates, such ashydrates, and further include both stoichiometric solvates andnon-stoichiometric solvates. In certain instances the solvate will becapable of isolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. ‘Solvate’encompasses both solution-phase and isolable solvates. Representativesolvates include hydrates, ethanolates and methanolates.

‘Subject’ includes humans. The terms ‘human’, ‘patient’ and ‘subject’are used interchangeably herein.

‘Therapeutically effective amount’ means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The ‘therapeutically effectiveamount’ can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringor developing a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset).

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting thedisease or reducing the manifestation, extent or severity of at leastone of the clinical symptoms thereof). In another embodiment ‘treating’or ‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, ‘treating’ or ‘treatment’ relates to slowingthe progression of the disease.

As used herein the term ‘inflammatory condition(s)’ refers to the groupof conditions including, rheumatoid arthritis, osteoarthritis, juvenileidiopathic arthritis, psoriasis, psoriatic arthritis, allergic airwaydisease (e.g. asthma, rhinitis), inflammatory bowel diseases (e.g.Crohn's disease, ulcerative colitis), endotoxin-driven disease states(e.g. complications after bypass surgery or chronic endotoxin statescontributing to e.g. chronic cardiac failure), and related diseasesinvolving cartilage, such as that of the joints. Particularly the termrefers to rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma) and inflammatory bowel diseases.

As used herein the term ‘autoimmune disease(s)’ refers to the group ofdiseases including obstructive airways disease, including conditionssuch as COPD, asthma (e.g. intrinsic asthma, extrinsic asthma, dustasthma, or infantile asthma) particularly chronic or inveterate asthma(for example late asthma and airway hyperreponsiveness), bronchitis(including bronchial asthma), systemic lupus erythematosus (SLE),cutaneous lupus erythematosus (CLE), multiple sclerosis, type I diabetesmellitus and complications associated therewith, atopic eczema (atopicdermatitis), contact dermatitis and further eczematous dermatitis,inflammatory bowel disease (e.g. Crohn's disease or ulcerative colitis),atherosclerosis and amyotrophic lateral sclerosis. Particularly the termrefers to COPD, asthma, type 1 diabetes mellitus and inflammatory boweldisease.

As used herein the term ‘proliferative disease(s)’ refers to conditionssuch as cancer (e.g. uterine leiomyosarcoma or prostate cancer),myeloproliferative disorders (e.g. polycythemia vera, essentialthrombocytosis and myelofibrosis), leukemia (e.g. acute myeloidleukaemia and acute lymphoblastic leukemia), multiple myeloma,psoriasis, restenosis, sclerodermitis or fibrosis. In particular theterm refers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term ‘cancer’ refers to a malignant or benign growthof cells in skin or in body organs, for example but without limitation,breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancertends to infiltrate into adjacent tissue and spread (metastasise) todistant organs, for example to bone, liver, lung or the brain. As usedherein the term cancer includes both metastatic tumour cell types, suchas but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such asbut not limited to, colorectal cancer, prostate cancer, small cell lungcancer and non-small cell lung cancer, breast cancer, pancreatic cancer,bladder cancer, renal cancer, gastric cancer, glioblastoma, primaryliver cancer, ovarian cancer, prostate cancer and uterineleiomyosarcoma.

As used herein the term ‘leukemia’ refers to neoplastic diseases of theblood and blood forming organs. Such diseases can cause bone marrow andimmune system dysfunction, which renders the host highly susceptible toinfection and bleeding. In particular the term leukemia refers to acutemyeloid leukaemia (AML) and acute lymphoblastic leukemia (ALL).

As used herein the term ‘transplantation rejection’ refers to the acuteor chronic rejection of cells, tissue or solid organ allo- or xenograftsof e.g. pancreatic islets, stem cells, bone marrow, skin, muscle,corneal tissue, neuronal tissue, heart, lung, combined heart-lung,kidney, liver, bowel, pancreas, trachea or oesophagus, orgraft-versus-host diseases.

As used herein the term ‘diseases involving impairment of cartilageturnover’ includes conditions such as osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,endemic forms of arthritis like osteoarthritis deformans endemica,Mseleni disease and Handigodu disease; degeneration resulting fromfibromyalgia, systemic lupus erythematosus, scleroderma and ankylosingspondylitis.

As used herein the term ‘congenital cartilage malformation(s)’ includesconditions such as hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

As used herein the term ‘disease(s) associated with hypersecretion ofIL6’ includes conditions such as Castleman's disease, multiple myeloma,psoriasis, Kaposi's sarcoma and/or mesangial proliferativeglomerulonephritis.

As used herein the term ‘disease(s) associated with hypersecretion ofinterferons’ includes conditions such as systemic and cutaneous lupuserythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome,psoriasis, rheumatoid arthritis.

‘Compound of the invention’, and equivalent expressions, are meant toembrace the compound according to Formula I or Formula II (as contextdictates) as hereinbefore described, which expression includes thepharmaceutically acceptable salts, and the solvates, e.g., hydrates, andthe solvates of the pharmaceutically acceptable salts where the contextso permits. Similarly, reference to intermediates, whether or not theythemselves are claimed, is meant to embrace their salts, and solvates,where the context so permits.

Other derivatives of the compound of the invention have activity in boththeir acid and acid derivative forms, but in the acid sensitive formoften offers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985).

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Further, compounds may beprepared that are substituted with positron emitting isotopes, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy.

All isotopic variants of the compound provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of a electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

The Invention

The present invention is based on the discovery that the compound of theinvention according to Formula I may be useful as a medicament. In aparticular aspect, the compound of the invention according to Formula Iis an inhibitor of JAK, and more particularly JAK1.

The present invention also provides methods for the production of thecompound of the invention according to Formula I, pharmaceuticalcompositions comprising the compound of the invention according toFormula I and methods for the prophylaxis and/or treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,allergy, transplant rejection, diseases involving impairment ofcartilage turnover, congenital cartilage malformations, and/or diseasesassociated with hypersecretion of IL6 or interferons, in particularrheumatoid arthritis, by administering the compound of the inventionaccording to Formula I.

Accordingly, in a first embodiment of the invention, a compound of theinvention according to Formula I is provided for use in medicine havinga Formula I:

In another embodiment, the compound of the invention may be prepared ina therapeutic combination, with a further therapeutically activeingredient. The further therapeutically active ingredient may be acompound for the treatment of the same conditions and diseases outlinedherein with respect to the compound of formula I. In a particularembodiment, the further therapeutically active ingredient may be acompound for the treatment of arthritis. In a most particular aspect,the further therapeutically active ingredient is a compound for thetreatment of rheumatoid arthritis.

In another embodiment of the invention, a compound of the invention,which is a JAK inhibitor, is provided for use in medicine having aFormula I:

The compound of the invention according to Formula I, surprisingly,exhibits in vivo in humans a very different profile from other animalspecies, which contrasts with in vitro predictions. Indeed, in vivo, ithas been demonstrated that in humans, the apparent terminal half life ofthe compound of the invention according to Formula I is significantlylonger than in the other animal species by at least 3 fold. This causesaccumulation in humans resulting in a maintained therapeutic effect overan extended period of time, thereby allowing once daily to once weeklydosing. Thus the compound of the invention according to Formula I mayprovide advantages including a low frequency dosage regimen and/orincreased patient compliance. In particular, the impact of nonadherence, if the patient misses a dose might be reduced.

In one embodiment the compound of the invention according to Formula Iis not an isotopic variant.

In one aspect the compound of the invention according to Formula I ispresent as the free base.

In one aspect the compound of the invention according to Formula I is apharmaceutically acceptable salt.

In one aspect the compound of the invention according to Formula I is asolvate of the compound of the invention according to Formula I.

In one aspect the compound of the invention according to Formula I is asolvate of a pharmaceutically acceptable salt of a compound of theinvention according to Formula I.

The compound of the invention according to Formula I is an inhibitor ofJAK. In particular the compound of the invention according to Formula Iis a potent inhibitor of JAK1, however it may inhibit JAK2, JAK3 andTYK2 with a lower potency.

In a further embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention according toFormula I, and a pharmaceutical carrier, excipient or diluent.

In yet a further embodiment, the pharmaceutical composition furthercomprises an additional therapeutic agent. In a particular aspect, theother compound is a compound for the treatment of arthritis. In a moreparticular aspect, the other compound is a compound for the treatment ofrheumatoid arthritis. In a most particular embodiment, the furthertherapeutic agent is a compound of the invention according to FormulaII:

Pharmaceutical Compositions

When employed as a pharmaceutical, the compound of the inventionaccording to Formula I is typically administered in the form of apharmaceutical composition. Such compositions can be prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound of the invention according to Formula I. Generally, thecompound of the invention according to Formula I is administered in apharmaceutically effective amount. The amount of the compound of theinvention according to Formula I actually administered will typically bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, the chosen route ofadministration, the actual compound of the invention according toFormula I administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

The pharmaceutical compositions of this invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intra-articular, intravenous, intramuscular, and intranasal. Dependingon the intended route of delivery, the compound of the inventionaccording to Formula I of this invention is preferably formulated aseither injectable or oral compositions or as salves, as lotions or aspatches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term ‘unit dosage forms’ refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention according to Formula I isusually a minor component (from about 0.1 to about 50% by weight orpreferably from about 1 to about 40% by weight) with the remainder beingvarious vehicles or carriers and processing aids helpful for forming thedesired dosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or non-aqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compound of the inventionsof a similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound of the invention according toFormula I in such compositions is typically a minor component, oftenbeing from about 0.05 to 10% by weight with the remainder being theinjectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as an ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

The compound of the invention according to Formula I can also beadministered by a transdermal device. Accordingly, transdermaladministration can be accomplished using a patch either of the reservoiror porous membrane type, or of a solid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17^(th) edition, 1985,Mack Publishing Company, Easton, Pa., which is incorporated herein byreference.

The compound of the invention according to Formula I can also beadministered in sustained release forms or from sustained release drugdelivery systems. A description of representative sustained releasematerials can be found in Remington's Pharmaceutical Sciences.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1 Tablets

The compound of the invention according to Formula I may be admixed as adry powder with a dry gelatin binder in an approximate 1:2 weight ratio.A minor amount of magnesium stearate may be added as a lubricant. Themixture may be formed into 240-270 mg tablets (80-90 mg of activecompound of the invention according to Formula I per tablet) in a tabletpress.

Formulation 2 Capsules

The compound of the invention according to Formula I may be admixed as adry powder with a starch diluent in an approximate 1:1 weight ratio. Themixture may be filled into 250 mg capsules (125 mg of active compound ofthe invention according to Formula I per capsule).

Formulation 3 Liquid

The compound of the invention according to Formula I (125 mg), may beadmixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultantmixture may be blended, passed through a No. 10 mesh U.S. sieve, andthen mixed with a previously made solution of microcrystalline celluloseand sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodiumbenzoate (10 mg), flavor, and color may be diluted with water and addedwith stirring. Sufficient water may then be added with stirring. Furthersufficient water may be then added to produce a total volume of 5 mL.

Formulation 4 Tablets

The compound of the invention according to Formula I may be admixed as adry powder with a dry gelatin binder in an approximate 1:2 weight ratio.A minor amount of magnesium stearate may be added as a lubricant. Themixture may be formed into 450-900 mg tablets (150-300 mg of activecompound of the invention according to Formula I) in a tablet press.

Formulation 5 Injection

The compound of the invention according to Formula I may be dissolved orsuspended in a buffered sterile saline injectable aqueous medium to aconcentration of approximately 5 mg/mL.

Formulation 6 Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted atabout 75° C. and then a mixture of the compound of the inventionaccording to Formula I (50 g) methylparaben (0.25 g), propylparaben(0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g)dissolved in water (about 370 g) may be added and the resulting mixturemay be stirred until it congeals.

Methods of Treatment

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention according toFormula I, for use in medicine. In a particular embodiment, the presentinvention provides a pharmaceutical composition comprising the compoundof the invention according to Formula I, for use in the prophylaxisand/or treatment of inflammatory conditions, autoimmune diseases,proliferative diseases, allergy, transplant rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6 orinterferons, in particular rheumatoid arthritis.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention according toFormula I, for use in the manufacture of a medicament for use in theprophylaxis and/or treatment of inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or interferons, in particular rheumatoid arthritis.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention according toFormula I, and another therapeutic agent. In a particular embodiment,the other therapeutic agent is an arthritis treatment agent. In a moreparticular embodiment, the other therapeutic agent is a rheumatoidarthritis treatment agent. In a most particular embodiment, the othertherapeutic agent is the compound according to Formula II.

In additional method of treatment aspects, this invention providesmethods of prophylaxis and/or treatment of a mammal susceptible to orafflicted with an inflammatory condition, which methods comprise theadministration of a therapeutically effective amount of the compound ofthe invention according to Formula I or one or more of thepharmaceutical compositions herein described for the treatment orprophylaxis of said condition. In a specific embodiment, theinflammatory condition is selected from rheumatoid arthritis,osteoarthritis, allergic airway disease (e.g. asthma) and inflammatorybowel diseases (e.g. Crohn's disease or ulcerative colitis).

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of an inflammatory condition. In aspecific embodiment, the inflammatory condition is selected fromrheumatoid arthritis, osteoarthritis, allergic airway disease (e.g.asthma) and inflammatory bowel diseases.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the manufacture of a medicament for the treatment or prophylaxis ofan inflammatory condition. In a specific embodiment, the inflammatorycondition is selected from rheumatoid arthritis, osteoarthritis,allergic airway disease (e.g. asthma) and inflammatory bowel diseases(e.g. Crohn's disease or ulcerative colitis).

In a method of treatment aspects, this invention provides methods oftreatment or prophylaxis of a mammal susceptible to or afflicted with anallergic reaction, which methods comprise the administration of atherapeutically effective amount of one or more of the pharmaceuticalcompositions or compound of the invention according to Formula I hereindescribed for the treatment or prophylaxis of said condition. In aspecific embodiment, the invention provides methods of treatment orprophylaxis of allergic airway disease, sinusitis, eczema and/or hives,food allergies or allergies to insect venom.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the treatment or prophylaxis of an allergic reaction. In a specificembodiment, the invention provides methods of treatment or prophylaxisof allergic airway disease, sinusitis, eczema and/or hives, foodallergies or allergies to insect venom.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the manufacture of a medicament for the treatment or prophylaxis ofan allergic reaction. In a specific embodiment, the invention providesmethods of treatment or prophylaxis of allergic airway disease,sinusitis, eczema and/or hives, food allergies or allergies to insectvenom.

In additional method of treatment aspects, this invention providesmethods of treatment or prophylaxis of a mammal susceptible to orafflicted with an autoimmune disease, which methods comprise theadministration of a therapeutically effective amount of one or more ofthe pharmaceutical compositions or compound of the invention accordingto Formula I herein described for the treatment or prophylaxis of saidcondition. In a specific embodiment, the autoimmune disease is selectedfrom rheumatoid arthritis, COPD, asthma, systemic lupus erythematosus,type I diabetes mellitus, psoriatic arthritis, ankylosing spondylitis,juvenile ideopathic arthritis, and inflammatory bowel disease (e.g.Crohn's disease or ulcerative colitis). In a more specific embodiment,the autoimmune disease is systemic lupus erythematosus. In yet anothermore specific embodiment, autoimmune disease is rheumatoid arthritis orpsoriatic arthritis. In yet a further specific embodiment, autoimmunedisease is inflammatory bowel disease (e.g. Crohn's disease orulcerative colitis).

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the treatment or prophylaxis of an autoimmune disease. In a specificembodiment, the autoimmune disease is selected from rheumatoidarthritis, COPD, asthma, systemic lupus erythematosus, type I diabetesmellitus, psoriatic arthritis, ankylosing spondylitis, juvenileideopathic arthritis, and inflammatory bowel disease (e.g. Crohn'sdisease or ulcerative colitis). In a more specific embodiment, theautoimmune disease is systemic lupus erythematosus. In yet another morespecific embodiment, autoimmune disease is rheumatoid arthritis orpsoriatic arthritis. In yet a further specific embodiment, autoimmunedisease is inflammatory bowel disease (e.g. Crohn's disease orulcerative colitis).

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the manufacture of a medicament for the treatment or prophylaxis ofan autoimmune disease. In a specific embodiment, the autoimmune diseaseis selected from rheumatoid arthritis, COPD, asthma, systemic lupuserythematosus, type I diabetes mellitus, psoriatic arthritis, ankylosingspondylitis, juvenile ideopathic arthritis, and inflammatory boweldisease (e.g. Crohn's disease or ulcerative colitis). In a more specificembodiment, the autoimmune disease is systemic lupus erythematosus. Inyet another more specific embodiment, autoimmune disease is rheumatoidarthritis or psoriatic arthritis. In yet a further specific embodiment,autoimmune disease is inflammatory bowel disease (e.g. Crohn's diseaseor ulcerative colitis).

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted witha proliferative disease, which methods comprise the administration of atherapeutically effective amount of one or more of the pharmaceuticalcompositions or compound of the invention according to Formula I hereindescribed for the treatment or prophylaxis of said condition. In aspecific embodiment, the proliferative disease is cancer (e.g. solidtumors such as uterine leiomyosarcoma or prostate cancer), cutaneousT-cell lymphoma, breast cancer, leukemia (e.g. AML, ALL or CLL),multiple myeloma and/or psoriasis. In a more specific embodiment, theproliferative disease is psoriasis.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the treatment or prophylaxis of a proliferative disease. In aspecific embodiment, the proliferative disease is cancer (e.g. solidtumors such as uterine leiomyosarcoma or prostate cancer), cutaneousT-cell lymphoma, breast cancer, leukemia (e.g. AML, ALL or CLL),multiple myeloma and/or psoriasis. In a more specific embodiment, theproliferative disease is psoriasis.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I, for usein the manufacture of a medicament for the treatment or prophylaxis of aproliferative disease. In a specific embodiment, the proliferativedisease is cancer (e.g. solid tumors such as uterine leiomyosarcoma orprostate cancer), cutaneous T-cell lymphoma, breast cancer, leukemia(e.g. AML, ALL or CLL), multiple myeloma and/or psoriasis. In a morespecific embodiment, the proliferative disease is psoriasis.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withtransplant rejection, which methods comprise the administration of atherapeutically effective amount of one or more of the pharmaceuticalcompositions, therapeutic combinations or compound of the inventionaccording to Formula I herein described for the treatment or prophylaxisof said condition. In a specific embodiment, the invention providesmethods of treatment or prophylaxis of organ transplant rejection.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of transplant rejection. In a specificembodiment, the invention provides methods of treatment or prophylaxisof organ transplant rejection.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the manufacture of a medicament for the treatment or prophylaxis oftransplant rejection. In a specific embodiment, the invention providesmethods of treatment or prophylaxis of organ transplant rejection.

In a method of treatment aspect, this invention provides a method oftreatment or prophylaxis in a mammal susceptible to or afflicted withdiseases involving impairment of cartilage turnover, which methodscomprise the administration of a therapeutically effective amount of oneor more of the pharmaceutical compositions or compound of the inventionaccording to Formula I herein described for the treatment or prophylaxisof said condition.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of diseases involving impairment ofcartilage turnover.

The present invention also provides a method of treatment or prophylaxisof congenital cartilage malformations, which methods comprise theadministration of a therapeutically effective amount of one or more ofthe pharmaceutical compositions or compound of the invention accordingto Formula I herein described for the treatment or prophylaxis of saidcondition.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of congenital cartilage malformations.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withdiseases associated with hypersecretion of IL6, which methods comprisethe administration of a therapeutically effective amount of one or moreof the pharmaceutical compositions or compound of the inventionaccording to Formula I herein described for the treatment or prophylaxisof said condition. In a specific embodiment, the disease associated withhypersecretion of IL6, is selected from Castleman's disease andmesangial proliferative glomerulonephritis.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of diseases associated withhypersecretion of IL6. In a specific embodiment, the disease associatedwith hypersecretion of IL6, is selected from Castleman's disease andmesangial proliferative glomerulonephritis.

In further method of treatment aspects, this invention provides methodsof treatment or prophylaxis of a mammal susceptible to or afflicted withdiseases associated with hypersecretion of interferons, which methodscomprise the administration of a therapeutically effective amount of oneor more of the pharmaceutical compositions or compound of the inventionaccording to Formula I herein described for the treatment or prophylaxisof said condition. In a specific embodiment, the disease associated withhypersecretion of interferon, is selected from systemic and cutaneouslupus erythematosus, lupus nephritis, dermatomyositis, Sjogren'ssyndrome, psoriasis, and rheumatoid arthritis.

In another aspect the present invention provides the compound of theinvention according to Formula I or a pharmaceutical compositioncomprising the compound of the invention according to Formula I for usein the treatment or prophylaxis of diseases associated withhypersecretion of interferons. In a specific embodiment, the diseaseassociated with hypersecretion of interferon, is selected from systemicand cutaneous lupus erythematosus, lupus nephritis, dermatomyositis,Sjogren's syndrome, psoriasis, and rheumatoid arthritis.

A particular regimen of the present method comprises the administrationto a subject suffering from a disease involving inflammation, inparticular rheumatoid arthritis, osteoarthritis, allergic airway disease(e.g. asthma) and/or inflammatory bowel diseases, of a therapeuticallyeffective amount of the compound of the invention according to Formula Ifor a period of time sufficient to reduce the level of inflammation inthe subject, and preferably terminate the processes responsible for saidinflammation. A special embodiment of the method comprises administeringof a therapeutically effective amount of the compound of the inventionaccording to Formula I to a subject patient suffering from orsusceptible to the development of rheumatoid arthritis, for a period oftime sufficient to reduce or prevent, respectively, inflammation in thejoints of said patient, and preferably terminate, the processesresponsible for said inflammation.

A further particular regimen of the present method comprises theadministration to a subject suffering from a disease or conditioncharacterized by cartilage or joint degradation (e.g. rheumatoidarthritis and/or osteoarthritis) of a therapeutically effective amountof the compound of the invention according to Formula I for a period oftime sufficient to reduce and preferably terminate the self-perpetuatingprocesses responsible for said degradation. A particular embodiment ofthe method comprises administering of a therapeutically effective amountof the compound of the invention according to Formula I to a subjectpatient suffering from or susceptible to the development ofosteoarthritis, for a period of time sufficient to reduce or prevent,respectively, cartilage degradation in the joints of said patient, andpreferably terminate, the self-perpetuating processes responsible forsaid degradation. In a particular embodiment said compound of theinvention according to Formula I may exhibit cartilage anabolic and/oranti-catabolic properties.

In one aspect, the present invention provides a compound of theinvention according to Formula I for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, inparticular rheumatoid arthritis, wherein the compound is administered inone to four (1-4) regular doses daily and especially one to three (1-3)regular doses daily, typically one to two (1-2) regular doses daily, andmost typically one (1) regular dose daily.

In another aspect, the present invention provides a compound of theinvention according to Formula I for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, inparticular rheumatoid arthritis, wherein the compound is administered inone to thirteen (1-13) regular doses in a two-week period. In a specificembodiment, the compound according to Formula I is administered in oneto twelve (1-12), one to ten (1-10) or two-seven (2-7) regular doses ina two week period. In a specific embodiment the compound of theinvention according to Formula I is administered on a once-weekly basis.

In a further aspect, the present invention provides a compound of theinvention according to Formula II for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, inparticular rheumatoid arthritis, wherein the compound is administered inone to thirteen (1-13) regular doses in a two-week period. In a specificembodiment, the compound according to Formula II is administered in oneto twelve (1-12), one to ten (1-10) or two-seven (2-7) regular doses ina two week period. In a specific embodiment the compound of theinvention according to Formula II is administered on a once-weeklybasis.

In yet a further aspect, the present invention provides a combination ofa compound of the invention according to Formula I and a compound of theinvention according to Formula II for use in the prophylaxis and/ortreatment of inflammatory conditions, autoimmune diseases, proliferativediseases, allergy, transplant rejection, diseases involving impairmentof cartilage turnover, congenital cartilage malformations, and/ordiseases associated with hypersecretion of IL6 or interferons, inparticular rheumatoid arthritis, wherein the compound is administered inone to four (1-4) regular doses daily and especially one to three (1-3)regular doses daily, typically one to two (1-2) regular doses daily, andmost typically one (1) regular dose daily.

In yet a another further aspect, the present invention provides acombination of a compound of the invention according to Formula I and acompound of the invention according to Formula II for use in theprophylaxis and/or treatment of inflammatory conditions, autoimmunediseases, proliferative diseases, allergy, transplant rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6 or interferons, in particular rheumatoid arthritis, wherein thecompound is administered in one to thirteen (1-13) regular doses in atwo-week period. In a specific embodiment, a combination of a compoundof the invention according to Formula I and a compound of the inventionaccording to Formula II is administered in one to twelve (1-12), one toten (1-10) or two-seven (2-7) regular doses in a two week period. In aspecific embodiment a combination of a compound of the inventionaccording to Formula I and a compound of the invention according toFormula II is administered on a once-weekly basis.

Injection dose levels range from about 0.1 mg/kg/h to at least 10mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h.A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more mayalso be administered to achieve adequate steady state levels. Themaximum total dose is not expected to exceed about 1 g/day for a 40 to80 kg human patient.

For the prophylaxis and/or treatment of long-term conditions, such asdegenerative conditions, the regimen for treatment usually stretchesover many months or years so oral dosing is preferred for patientconvenience and tolerance. With oral dosing, one to four (1-4) regulardoses daily, especially one to three (1-3) regular doses daily,typically one to two (1-2) regular doses daily, and most typically one(1) regular dose daily are representative regimens. Alternatively forlong lasting effect drugs, with oral dosing, once every other week, onceweekly, and once a day are representative regimens. In particular,dosage regimen can be every 1-14 days, more particularly 1-10 days, evenmore particularly 1-7 days, and most particularly 1-3 days.

Using these dosing patterns, each dose provides from about 1 to about1000 mg of the compound of the invention according to Formula I, withparticular doses each providing from about 10 to about 500 mg andespecially about 30 to about 250 mg.

Using these dosing patterns, each dose provides from about 1 to about1000 mg of the compound of the invention according to Formula II, withparticular doses each providing from about 10 to about 500 mg andespecially about 30 to about 250 mg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of a condition, the compound of theinvention according to Formula I will be administered to a patient atrisk for developing the condition, typically on the advice and under thesupervision of a physician, at the dosage levels described above.Patients at risk for developing a particular condition generally includethose that have a family history of the condition, or those who havebeen identified by genetic testing or screening to be particularlysusceptible to developing the condition.

The compound of the invention according to Formula I can be administeredas the sole active agent or it can be administered in combination withother therapeutic agents, including other compound of the inventionsthat demonstrate the same or a similar therapeutic activity and that aredetermined to safe and efficacious for such combined administration. Ina specific embodiment, co-administration of two (or more) agents allowsfor significantly lower doses of each to be used, thereby reducing theside effects seen.

In one embodiment, the compound of the invention according to Formula Ior a pharmaceutical composition comprising the compound of the inventionaccording to Formula I is administered as a medicament. In a specificembodiment, said pharmaceutical composition additionally comprises afurther active ingredient.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of a disease involving inflammation; particularagents include, but are not limited to, immunoregulatory agents e.g.azathioprine, corticosteroids (e.g. prednisolone or dexamethasone),cyclophosphamide, cyclosporin A, tacrolimus, Mycophenolate Mofetil,muromonab-CD3 (OKT3, e.g. Orthocolone®), ATG, aspirin, acetaminophen,ibuprofen, naproxen, and piroxicam.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of arthritis (e.g. rheumatoid arthritis); particularagents include but are not limited to analgesics, non-steroidalanti-inflammatory drugs (NSAIDS), steroids, synthetic DMARDS (forexample but without limitation methotrexate, leflunomide, sulfasalazine,auranofin, sodium aurothiomalate, penicillamine, chloroquine,hydroxychloroquine, azathioprine, tofacitinib, baricitinib,fostamatinib, and cyclosporin), and biological DMARDS (for example butwithout limitation Infliximab, Etanercept, Adalimumab, Rituximab, andAbatacept).

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of proliferative disorders; particular agents includebut are not limited to: methotrexate, leukovorin, adriamycin,prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel,docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin,tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin,anti-HER2 monoclonal antibody (e.g. Herceptin™), capecitabine,raloxifene hydrochloride, EGFR inhibitors (e.g. Lressa®, Tarceva™,Erbitux™), VEGF inhibitors (e.g. Avastin™), proteasome inhibitors (e.g.Velcade™), Glivec® and hsp90 inhibitors (e.g. 17-AAG). Additionally, thecompound of the invention according to Formula I may be administered incombination with other therapies including, but not limited to,radiotherapy or surgery. In a specific embodiment the proliferativedisorder is selected from cancer, myeloproliferative disease orleukaemia.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of autoimmune diseases, particular agents include butare not limited to: glucocorticoids, cytostatic agents (e.g. purineanalogs), alkylating agents, (e.g. nitrogen mustards (cyclophosphamide),nitrosoureas, platinum compound of the inventions, and others),antimetabolites (e.g. methotrexate, azathioprine and mercaptopurine),cytotoxic antibiotics (e.g. dactinomycin anthracyclines, mitomycin C,bleomycin, and mithramycin), antibodies (e.g. anti-CD20, anti-CD25 oranti-CD3 (OTK3) monoclonal antibodies, Atgam® and Thymoglobuline®),cyclosporin, tacrolimus, rapamycin (sirolimus), interferons (e.g.IFN-β), TNF binding proteins (e.g. infliximab, etanercept, oradalimumab), mycophenolate, Fingolimod and Myriocin.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of transplant rejection, particular agents includebut are not limited to: calcineurin inhibitors (e.g. cyclosporin ortacrolimus (FK506)), mTOR inhibitors (e.g. sirolimus, everolimus),anti-proliferatives (e.g. azathioprine, mycophenolic acid),corticosteroids (e.g. prednisolone, hydrocortisone), Antibodies (e.g.monoclonal anti-IL-2Rα receptor antibodies, basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g. anti-thymocyte globulin (ATG),anti-lymphocyte globulin (ALG)).

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of asthma and/or rhinitis and/or COPD, particularagents include but are not limited to: beta2-adrenoceptor agonists (e.g.salbutamol, levalbuterol, terbutaline and bitolterol), epinephrine(inhaled or tablets), anticholinergics (e.g. ipratropium bromide),glucocorticoids (oral or inhaled) Long-acting β2-agonists (e.g.salmeterol, formoterol, bambuterol, and sustained-release oralalbuterol), combinations of inhaled steroids and long-actingbronchodilators (e.g. fluticasone/salmeterol, budesonide/formoterol),leukotriene antagonists and synthesis inhibitors (e.g. montelukast,zafirlukast and zileuton), inhibitors of mediator release (e.g.cromoglycate and ketotifen), biological regulators of IgE response (e.g.omalizumab), antihistamines (e.g. ceterizine, cinnarizine, fexofenadine)and vasoconstrictors (e.g. oxymethazoline, xylomethazoline, nafazolineand tramazoline).

Additionally, the compound of the invention according to Formula I maybe administered in combination with emergency therapies for asthmaand/or COPD, such therapies include oxygen or heliox administration,nebulized salbutamol or terbutaline (optionally combined with ananticholinergic (e.g. ipratropium), systemic steroids (oral orintravenous, e.g. prednisone, prednisolone, methylprednisolone,dexamethasone, or hydrocortisone), intravenous salbutamol, non-specificbeta-agonists, injected or inhaled (e.g. epinephrine, isoetharine,isoproterenol, metaproterenol), anticholinergics (IV or nebulized, e.g.glycopyrrolate, atropine, ipratropium), methylxanthines (theophylline,aminophylline, bamiphylline), inhalation anesthetics that have abronchodilatory effect (e.g. isoflurane, halothane, enflurane), ketamineand intravenous magnesium sulfate.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of inflammatory bowel disease (IBD), particularagents include but are not limited to: glucocorticoids (e.g. prednisone,budesonide) synthetic disease modifying, immunomodulatory agents (e.g.methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine,6-mercaptopurine and cyclosporin) and biological disease modifying,immunomodulatory agents (infliximab, adalimumab, rituximab, andabatacept).

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of SLE, particular agents include but are not limitedto: Disease-modifying antirheumatic drugs (DMARDs) such as antimalarials(e.g. plaquenil, hydroxychloroquine), immunosuppressants (e.g.methotrexate and azathioprine), cyclophosphamide and mycophenolic acid;immunosuppressive drugs and analgesics, such as nonsteroidalanti-inflammatory drugs, opiates (e.g. dextropropoxyphene andco-codamol), opioids (e.g. hydrocodone, oxycodone, MS Contin, ormethadone) and the fentanyl duragesic transdermal patch.

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of psoriasis, particular agents include but are notlimited to: topical treatments such as bath solutions, moisturizers,medicated creams and ointments containing coal tar, dithranol(anthralin), corticosteroids like desoximetasone (Topicort™),fluocinonide, vitamin D3 analogues (for example, calcipotriol), Arganoiland retinoids (etretinate, acitretin, tazarotene), systemictreatments such as methotrexate, cyclosporine, retinoids, tioguanine,hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine,tacrolimus, fumaric acid esters or biologics such as Amevive™, Enbrel™,Humira™, Remicade™, Raptiva™ and ustekinumab (a IL-12 and IL-23blocker). Additionally, the compound of the invention according toFormula I may be administered in combination with other therapiesincluding, but not limited to phototherapy, or photochemotherapy (e.g.psoralen and ultraviolet A phototherapy (PUVA)).

In one embodiment, the compound of the invention according to Formula Iis co-administered with another therapeutic agent for the treatmentand/or prophylaxis of allergic reaction, particular agents include butare not limited to: antihistamines (e.g. cetirizine, diphenhydramine,fexofenadine, levocetirizine), glucocorticoids (e.g. prednisone,betamethasone, beclomethasone, dexamethasone), epinephrine, theophyllineor anti-leukotrienes (e.g. montelukast or zafirlukast),anti-cholinergics and decongestants.

By co-administration is included any means of delivering two or moretherapeutic agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation, i.e. as asingle pharmaceutical composition, this is not essential. The agents maybe administered in different formulations and at different times.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound of the invention according toFormula I, and the compound according to Formula II, wherein the ratioof the compound of the invention according to Formula I/Formula II isfrom 1/5 to 1/20. In a particular embodiment, the ratio is from 1/5 to1/10.

General Synthetic Procedures General

The compound of the invention according to Formula I can be preparedfrom readily available starting materials using the following generalmethods and procedures. It will be appreciated that where typical orpreferred process conditions (i.e. reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

The following methods are presented with details as to the preparationof the compound of the invention according to Formula I as definedhereinabove and the comparative examples. The compound of the inventionaccording to Formula I may be prepared from known or commerciallyavailable starting materials and reagents by one skilled in the art oforganic synthesis.

All reagents were of commercial grade and were used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents were used for reactions conducted under inertatmosphere. Reagent grade solvents were used in all other cases, unlessotherwise specified. Column chromatography was performed on silica gel60 (35-70 μm). Thin layer chromatography was carried out usingpre-coated silica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectrawere recorded on a Bruker DPX 400 NMR spectrometer (400 MHz). Chemicalshifts (δ) for ¹H NMR spectra are reported in parts per million (ppm)relative to tetramethylsilane (δ 0.00) or the appropriate residualsolvent peak, i.e. CHCl₃ (δ 7.27), as internal reference. Multiplicitiesare given as singlet (s), doublet (d), triplet (t), quartet (q),multiplet (m) and broad (br). Coupling constants (J) are given in Hz.Electrospray MS spectra were obtained on a Micromass platform LC/MSspectrometer. Columns Used for LCMS analysis: Hichrom, KromasilEternity, 2.5 μm C₁₈, 150×4.6 mm, Waters Xbridge 5 μm C₁₈ (2), 250×4.6mm (ref 86003117), Waters Xterra MS 5 μm C₁₈, 100×4.6 mm (Plus guardcartridge) (ref 186000486), Gemini-NX 3 μm C₁₈ 100×3.0 mm (ref00D-4453-Y0), Phenomenex Luna 5 μm C₁₈ (2), 100×4.6 mm. (Plus guardcartridge) (ref 00D-4252-E0), Kinetix fused core 2.7 μm C₁₈ 100×4.6 mm(ref 00D-4462-E0), Supelco, Ascentis® Express C₁₈ (ref 53829-U), orHichrom Halo C₁₈, 2.7 μm C₁₈, 150×4.6 mm (ref 92814-702). LC-MS wererecorded on a Waters Micromass ZQ coupled to a HPLC Waters 2795,equipped with a UV detector Waters 2996. LC were also run on a HPLCAgilent 1100 coupled to a UV detector Agilent G1315A. Preparative HPLC:Waters XBridge Prep C₁₈ 5 μm ODB 19 mm ID×100 mm L (Part No. 186002978).All the methods are using MeCN/H₂O gradients. H₂O contains either 0.1%TFA or 0.1% NH₃.

List of abbreviations used in the experimental section:

AUC Area Under the Curve APC Adenomatous Polyposis Coli DCMDichloromethane Cat. Catalytic amount TFA Trifluoroacetic acid THFTetrahydrofuran NMR Nuclear Magnetic Resonnance DMSO DimethylsulfoxideLC-MS Liquid Chromatography- Mass Spectrometry ppm part-per-millionEtOAc ethyl acetate Rt retention time s singlet br s broad singlet mmultiplet min minute mL milliliter μL microliter g gram mg milligramDiPEA N,N-diisopropylethylamine MeCN Acetonitrile DMFN,N-dimethylformamide PdCl₂dppf [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) MMP Matrix Metallo Proteinase RNA Ribonucleic acidAPMA 4-aminophenylmercuric acetate FBS Fetal bovine serum cDNA copydeoxyribonucleic acid h hour FITC Fluorescein Isothiocyanate mmolmillimoles QD Quaque Die (once daily dosing) BID Bis in Die (twicedaily) Rel. Relative HPLC High pressure liquid chromatography

Synthetic Preparation of the Compound of the Invention Example 1Synthesis of the Compounds 1.1. Route 1 1.1.1. Synthesis of5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (Intermediate 3)

1.1.1.1. 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (2)

To a solution of 2-amino-6-bromopyridine (1) (253.8 g, 1.467 mol) in DCM(2.5 L) cooled to 5° C. was added ethoxycarbonyl isothiocyanate (173.0mL, 1.467 mol) dropwise over 15 min. The reaction mixture was thenallowed to warm to room temp. (20° C.) and stirred for 16 h Evaporationin vacuo gave a solid which was collected by filtration, thoroughlywashed with petrol (3×600 mL) and air-dried to afford (2). The thioureawas used as such in the next step without any purification.

¹H (400 MHz, CDCl₃) δ 12.03 (1H, br s, NH), 8.81 (1H, d, J 7.8 Hz, H-3),8.15 (1H, br s, NH), 7.60 (1H, t, J 8.0 Hz, H-4), 7.32 (1H, dd, J 7.7and 0.6 Hz, H-5), 4.31 (2H, q, J 7.1 Hz, CH₂), 1.35 (3H, t, J 7.1 Hz,CH₃).

1.1.1.2. 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (3)

To a suspension of hydroxylamine hydrochloride (101.8 g, 1.465 mol) inEtOH/MeOH (1:1, 900 mL) was added N,N-diisopropylethylamine (145.3 mL,0.879 mol) and the mixture was stirred at room temp. (20° C.) for 1 h.1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (2) (89.0 g, 0.293 mol)was then added and the mixture slowly heated to reflux (Note: bleachscrubber was required to quench H₂S evolved). After 3 h at reflux, themixture was allowed to cool and filtered to collect the precipitatedsolid. Further product was collected by evaporation in vacuo of thefiltrate, addition of H₂O (250 mL) and filtration. The combined solidswere washed successively with H₂O (250 mL), EtOH/MeOH (1:1, 250 mL) andEt₂O (250 mL) then dried in vacuo to afford the triazolopyridinederivative (3) as a solid. The compound was used as such in the nextstep without any purification.

¹H (400 MHz, DMSO-d₆) δ 7.43-7.34 (2H, m, 2× aromatic-H), 7.24 (1H, dd,J 6.8 and 1.8 Hz, aromatic-H), 6.30 (2H, br, NH₂); m/z 213/215 (1:1,M+H⁺, 100%).

1.1.2. Synthesis of4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-thiomorpholine-1,1-dioxide(Intermediate 4)

2-(4-Bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1 eq)and DIPEA (2 eq) were dissolved in DCM/MeOH (5:1 v:v) under N₂ andthiomorpholine 1,1-dioxide (2 eq) was added portion wise. The resultingsolution was stirred at room temperature for 16 h. After this time, thereaction was complete. The solvent was evaporated. The compound wasextracted with EtOAc and water, washed with brine and dried overanhydrous MgSO₄. Organic layers were filtered and evaporated. The finalcompound was isolated without further purification.

1.1.3. Synthesis of5-[4-(1,1-Dioxothiomorpholin-4-ylmethyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine(Formula I)

4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-thiomorpholine-1,1-dioxide(1.1 eq.) was added to a solution of5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (4:1). K₂CO₃ (2 eq.) andPdCl₂dppf (0.03 eq.) were added to the solution. The resulting mixturewas then heated in an oil bath at 90° C. for 16 h under N₂. Water wasadded and the solution was extracted with ethyl acetate. The organiclayers were dried over anhydrous MgSO₄ and evaporated in vacuo. Thefinal compound was obtained after purification by flash chromatography.

¹H (400 MHz, CDCl₃) δ 7.94-7.92 (d, 2H), 7.52-7.48 (m, 3H), 7.37-7.34(m, 1H), 7.02-7.00 (m, 1H), 6.00 (d, 2H), 3.76 (d, 2H), 3.15-3.13 (m,4H), 2.93-2.91 (m, 4H).

m/z 358.2 (M+H⁺, 100%).

1.2. Route 2 1.2.1. Cyclopropanecarboxylic acid{5-[4-(1,1-dioxo-thiomorpholin-4-ylmethyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide(Formula II)

The compound according to Formula II may be synthesized according to theprocedure described in WO 2010/149769.

1.2.2. Synthesis of5-[4-(1,1-Dioxothiomorpholin-4-ylmethyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine(Formula I)

The compound according to Formula I can also be produced by hydrolysisof the compound according to Formula II:

Hydrochloric acid 30% aq (12.06 kg; 3.9 rel. volumes) was added to aslurry of the compound according to Formula II (3.45 kg; 1.0 equiv.) indemineralized water (10.0 kg; 3.0 rel. volumes). Subsequently, a linerinse was performed with demineralized water (3.4 kg; 1.0 rel. volumes).The reaction mixture was heated to 80±5° C. for 14.5 h. After completionof the reaction (conversion≧99%), the reaction mixture was cooled to20±5° C. The reaction mixture was diluted with demineralized water (6.8kg; 2.0 rel. volumes) and sodium hydroxide 33% aq (9.52 kg; 3.7 relvolumes) was dosed at such a rate that the temperature of the reactorcontents remained below 35° C. An additional amount of sodium hydroxide33% aq (2.55 kg; 1.0 rel. volumes) was needed to get the pH≧10. Theproduct was filtered off, washed twice with demineralized water (1.5rel. volumes) and dried under vacuum for 1 h, thus yielding the crudecompound according to Formula I.

The crude compound according to Formula I (5.70 kg) was re-slurried indemineralized water (23.0 kg; 8.5 rel. volumes). Hydrochloric acid 30%aq (1.65 kg; 0.7 rel. volumes) and demineralized water (4.3 kg; 1.6 rel.volumes) were added and the reaction mixture was stirred at 20±5° C. for45 min. As the compound according to Formula I was not dissolvedcompletely, the reaction mixture was stirred at 455° C. for 1 h. Thereaction mixture was filtered and the residue was washed withdemineralized water (2.0 kg 0.75 rel. volumes). Sodium hydroxide 33% aq(1.12 kg; 0.6 rel volumes) was added to the filtrate. An additionalamount of sodium hydroxide 33% aq (1.01 kg) was needed to get the pH≧10.The resulting reaction mixture was stirred at 20±5° C. for about 3 h.The product was filtered off, washed twice with demineralized water (4.1kg; 1.5 rel. volumes), and twice with methyl tert-butyl ether (MTBE; 3.0kg; 1.5 rel. volumes) and dried under vacuum for 15.5 h on the filter.The product was further dried in a vacuum oven at 40±5° C. for 202 h,thus affording the desired compound according to Formula I.

Biological Examples Example 2 In-Vitro Assays 2.1. JAK1 Inhibition Assay2.1.1. JAK1 Assay polyGT Substrate

Recombinant human JAK1 catalytic domain (amino acids 866-1154; catalognumber PV4774) was purchased from Invitrogen. 25 ng of JAK1 wasincubated with 6.25 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (15 mM Hepes pH7.5, 0.01% Tween20, 10 mM MgCl₂, 2μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP (Perkin Elmer, catalognumber NEG602K001MC) final concentrations) with or without 5 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 75 min at 30° C., reactions were stopped by adding 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionwas transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates was scaled. 40μL/well of Microscint-20 was added, the top of the plates was sealed andreadout was performed using the Topcount (Perkin Elmer). Kinase activitywas calculated by subtracting counts per min (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity was determined as:

Percentage inhibition−((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK1 assay and the calculationof the IC₅₀ for the compound. The compound was tested at a concentrationof 20 μM followed by a 1/3 serial dilution, 8 points (20 μM-6.67 μM-2.22μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a final concentration of 1% DMSO.When compound potency increases, more dilutions were prepared and/or thetop concentration is lowered (e.g. 5 μM, 1 μM).

The activity of the compound according to Formula I against JAK1 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of 460.1, 586, 494.3, 758.2, and 432.7 nM (average 546.26nM).

2.1.2. JAK1 Ulight-JAK1 Peptide Assay

Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalognumber PV4774) was purchased from Invitrogen. 1 ng of JAK1 was incubatedwith 20 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalog numberTRF0121) in kinase reaction buffer (15 mM MOPS pH6.8, 0.01% Brij-35, 5mM MgCl₂, 2 mM DTT, 7 μM ATP) with or without 4 μL containing testcompound or vehicle (DMSO, 1% final concentration), in a total volume of20 μL, in a white 384 Opti plate (Perkin Elmer, catalog number 6007290).After 60 min at room temperature, reactions were stopped by adding 20μL/well of detection mixture (1× detection buffer (Perkin Elmer, catalognumber CR97-100C), 0.5 nM Europium-anti-phosphotyrosine (PT66) (PerkinElmer, catalog number AD0068), 10 mM EDTA). Readout was performed usingthe Envision with excitation at 320 nm and measuring emission at 615 nm(Perkin Elmer). Kinase activity was calculated by subtracting relativefluorescence units (RFU) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from RFU obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:

Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

A dose dilution series was prepared for the compound enabling thetesting of dose-response effects in the JAK1 assay and the calculationof the IC₅₀ for the compound. The compound was routinely tested at aconcentration of 20 μM followed by a 1/5 serial dilution, 10 points in afinal concentration of 1% DMSO. When compound potency increases, moredilutions were prepared and/or the top concentration is lowered (e.g. 5μM, 1 μM). The data were expressed as the average IC₅₀ from theassays±standard error of the mean.

The activity of the compound according to Formula I against JAK1 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of 346.8, 714.3, 166.6, 103.3, 187.2, 582.3, 295.3, 241.7,159.2, 355.3, and 221.6 nM (average=307 nM).

2.1.3. JAK1 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 1 ng ofJAK1 (Invitrogen, PV4774) is used in the assay. The substrate is 20 nMUlight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121). The reaction isperformed in 15 mM MOPS pH 6.8, 0.01%, Brij-35, 2 mM DTT, 5 mM MgCl2with varying concentrations of ATP and compound. Phosphorylatedsubstrate is measured using an Europium-labeled anti-phosphotyrosineantibody PT66 (Perkin Elmer, AD0068) as described in 1.1.2. Readout isperformed on the envision (Perkin Elmer) with excitation at 320 nm andemission followed at 615 nm and 665 nm.

2.2. JAK2 Inhibition Assay 2.2.1. JAK2 Assay polyGT Substrate

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalognumber PV4210) was purchased from Invitrogen. 0.05 mU of JAK2 wasincubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (10 mM MOPS pH 7.5, 0.5 mM EDTA, 0.01% Brij-35, 1mM DTT, 15 mM MgAc, 1 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP(Perkin Elmer, catalog number NEG602K001MC) final concentrations) withor without 5 μL containing test compound or vehicle (DMSO, 1% finalconcentration), in a total volume of 25 μL, in a polypropylene 96-wellplate (Greiner, V-bottom). After 90 min at 30° C., reactions werestopped by adding of 25 μL/well of 150 mM phosphoric acid. All of theterminated kinase reaction was transferred to prewashed (75 mMphosphoric acid) 96 well filter plates (Perkin Elmer catalog number6005177) using a cell harvester (Perkin Elmer). Plates were washed 6times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates was sealed. 40 μL/well of Microscint-20 was added,the top of the plates was sealed and readout was performed using theTopcount (Perkin Elmer). Kinase activity was calculated by subtractingcounts per min (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK2 assay and the calculationof the IC₅₀ for each compound. The compound was tested at aconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.671 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increases,more dilutions were prepared and/or the top concentration is lowered(e.g. 5 μM, 1 μM).

The activity of the compound according to Formula I against JAK1 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of 566.9, 365.5, 256.4, 915.1, and 1017 nM (average=624 nM).

2.2.2. JAK2 Ulight-JAK1 Peptide Assay

Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalognumber PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 wasincubated with 25 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalognumber TRF0121) in kinase reaction buffer (25 mM HEPES pH7.0, 0.01%Triton X-100, 7.5 mM MgCl₂, 2 mM DTT, 7.5 μM ATP) with or without 4 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 20 μL, in a white 384 Opti plate (Perkin Elmer, catalognumber 6007290). After 60 min at room temperature, reactions werestopped by adding 20 μL/well of detection mixture (1× detection buffer(Perkin Elmer, catalog number CR97-100C), 0.5 nMEuropium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog numberAD0068), 10 mM EDTA). Readout was performed using the Envision withexcitation at 320 nm and measuring emission at 615 nm (Perkin Elmer).Kinase activity was calculated by subtracting relative fluorescenceunits (RFU) obtained in the presence of a positive control inhibitor (10μM staurosporine) from RFU obtained in the presence of vehicle. Theability of a test compound to inhibit this activity was determined as:

Percentage inhibition−((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for compound enabling the testing ofdose-response effects in the JAK2 assay and the calculation of the IC₅₀for the compound. The compound was tested at concentration of 20 μMfollowed by a 1/5 serial dilution, 10 points in a final concentration of1% DMSO. When compound potency increases, more dilutions were preparedand/or the top concentration was lowered (e.g. 5 μM, 1 μM). The datawere expressed as the average IC₅₀ from the assays±standard error of themean.

The activity of the compound according to Formula I against JAK2 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of 1031, 351.2, 137.5, 367.2, 310.2, 729.2, 151.7, 203.0,168.0, and 517.0 (average=397 nM).

2.2.3. JAK2 Kd/Ki Determination Assay 2.2.3.1. JAK2 Ki DeterminationAssay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 0.0125mU of JAK1 (Invitrogen, PV4210) is used in the assay. The substrate is25 nM Ulight-JAK-1 (Tyr1023) Peptide (Perkin Elmer, TRF0121). Thereaction is performed in 25 mM HEPES pH7.0, 0.01% Triton X-100, 7.5 mMMgCl₂, 2 mM DTT with varying concentrations of ATP and compound.Phosphorylated substrate is measured using a Europium-labeledanti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068) as describedin 1.2.2. Readout is performed on the envision (Perkin Elmer) withexcitation at 320 nm and emission followed at 615 nm and 665 nm.

2.2.3.2. JAK2 Kd Determination Assay

JAK2 (Invitrogen, PV4210) is used at a final concentration of 2.5 nM.The binding experiment is performed in 50 mM Hepes pH 7.5, 0.01%Brij-35, 10 mM MgCl₂, 1 mM EGTA using 25 nM kinase tracer 236(Invitrogen, PV5592) and 2 nM Europium-anti-GST (Invitrogen, PV5594)with varying compound concentrations. Detection of tracer is performedaccording to the manufacturer's procedure.

2.2.4. JAK3 Inhibition Assay

Recombinant human JAK3 catalytic domain (amino acids 795-1124; catalognumber 08-046) was purchased from Carna Biosciences. 0.5 ng JAK3 proteinwas incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275)in kinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 10 mM MgCl₂,2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mM b-glycerophosphate, 0.01% Triton X-100,1 μM non-radioactive ATP, 0.25 μCi ³³P-gamma-ATP (Perkin Elmer, catalognumber NEG602K001MC) final concentrations) with or without 5 μLcontaining test compound or vehicle (DMSO, 1% final concentration), in atotal volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 45 min at 30° C., reactions were stopped by adding 25μL/well of 150 mM phosphoric acid. All of the terminated kinase reactionwas transferred to prewashed (75 mM phosphoric acid) 96 well filterplates (Perkin Elmer catalog number 6005177) using a cell harvester(Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75mM phosphoric acid solution and the bottom of the plates was sealed. 40μL/well of Microscint-20 was added, the top of the plates was sealed andreadout was performed using the Topcount (Perkin Elmer). Kinase activitywas calculated by subtracting counts per min (cpm) obtained in thepresence of a positive control inhibitor (10 μM staurosporine) from cpmobtained in the presence of vehicle. The ability of a test compound toinhibit this activity was determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK3 assay and the calculationof the IC₅₀ for the compound. The compound was tested at concentrationof 20 μM followed by a 1/5 serial dilution, 10 points in a finalconcentration of 1% DMSO. When compound increased, more dilutions wereprepared and/or the top concentration was lowered (e.g. 5 μM, 1 μM).

The activity of the compound according to Formula I against JAK3 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of 2497, >4000, >4000, >3333, >3333,3939, >4000, >4000, >4000, 3201, and 3368 (average >3606 nM).

2.2.5. JAK3 Ki Determination Assay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK3(Carna Biosciences, 08-046) is used at a final concentration of 20ng/mL. The substrate is Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000(Sigma, P0275) The reaction is performed in 25 mM Tris pH 7.5, 0.01%Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mMb-glycerolphosphate, 10 mM MgCl₂ with varying concentrations of ATP andcompound and stopped by addition of 150 mM phosphoric acid. Measurementof incorporated phosphate into the substrate polyGT is done by loadingthe samples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated ³³P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer).

2.3. TYK2 Inhibition Assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalognumber 08-147) was purchased from Carna biosciences. 4 ng of TYK2 wasincubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) inkinase reaction buffer (25 mM Hepes pH 7.2, 50 mM NaCl, 0.5 mM EDTA, 1mM DTT, 5 mM MnCl₂, 10 mM MgCl₂, 0.1% Brij-35, 0.1 μM non-radioactiveATP, 0.125 μCi ³³P-gamma-ATP (Perkin Elmer, catalog number NEG602K001MC)final concentrations) with or without 5 μL containing test compound orvehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30° C.,reactions were stopped by adding 25 μL/well of 150 mM phosphoric acid.All of the terminated kinase reaction was transferred to prewashed (75mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number6005177) using a cell harvester (Perkin Elmer). Plates were washed 6times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates was sealed. 40 μL/well of Microscint-20 was added,the top of the plates was sealed and readout was performed using theTopcount (Perkin Elmer). Kinase activity was calculated by subtractingcounts per min (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity wasdetermined as:

Percentage inhibition−((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the TYK2 assay and the calculationof the IC₅₀ for each compound. Each compound was routinely tested atconcentration of 2 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions were prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM).

The activity of the compound according to Formula I against TYK2 wasdetermined in accordance with the assay described above, thus returningIC₅₀ values of >3333, >3333, >3333, 1973, 2121, 3852, 3819, and 2207(average >2996 nM).

2.3.1. TYK2 Kd/Ki Determination Assay 2.3.1.1. TYK2 Ki DeterminationAssay

For the determination of Ki, different amounts of compound are mixedwith the enzyme and the enzymatic reaction is followed as a function ofATP concentration. The Ki is determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). TYK2(Carna Biosciences, 08-147) is used at a final concentration of 160ng/mL. The substrate is Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000(Sigma, P0275) The reaction is performed in 25 mM Hepes pH 7.2, 50 mMNaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM MnCl₂, 10 mM MgCl₂, 0.1% Brij-35 withvarying concentrations of ATP and compound and stopped by addition of150 mM phosphoric acid. Measurement of incorporated phosphate into thesubstrate polyGT is done by loading the samples on a filter plate (usinga harvester, Perkin Elmer) and subsequent washing. Incorporated ³³P inpolyGT is measured in a Topcount scintillation counter after addition ofscintillation liquid to the filter plates (Perkin Elmer).

2.3.1.2. TYK2 Kd Determination Assay

TYK2 (Carna Biosciences, 08-147) is used at a final concentration of 50nM. The binding experiment is performed in 50 mM Hepes pH 7.5, 0.01%Brij-35, 10 mM MgCl₂, 1 mM EGTA using 15 nM kinase tracer 236(Invitrogen, PV5592) and 10 nM Europium-anti-GST (Invitrogen, PV5594)with varying compound concentrations. Detection of tracer is performedaccording to the manufacturers' procedure.

Example 3 Cellular Assays 3.1. JAK-STAT Signalling Assay

HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)containing 10% heat inactivated fetal calf serum, 100 U/mL penicillinand 100 μg/mL streptomycin. HeLa cells were used at 70% confluence fortransfection. 20,000 cells in 87 μL cell culture medium were transientlytransfected with 40 ng pSTAT1(2)-luciferase reporter (Panomics), 8 ng ofLacZ reporter as internal control reporter and 52 ng of pBSK using 0.32μL Jet-PEI (Polyplus) as transfection reagent per well in 96-well plateformat. After overnight incubation at 37° C., 5% CO₂, transfectionmedium was removed. 81 μL of DMEM+1.5% heat inactivated fetal calf serumwas added. 9 μL compound at 10× concentration was added for 60 min andthen 10 μL of human OSM (Peprotech) at 33 ng/mL final concentration.

The compound was tested in duplicate starting from 20 μM followed by a1/3 serial dilution, 8 doses in total (20 μM-6.6 μM-2.2 μM-740 nM-250nM-82 nM-27 nM-9 nM) in a final concentration of 0.2% DMSO.

After overnight incubation at 37° C., 5% CO₂ cells were lysed by adding100 μL lysis buffer/well (PBS, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 10%Trehalose, 0.05% Tergitol NP9, 0.3% BSA).

40 μL of cell lysate was used to read β-galactosidase activity by adding180 μL 11-Gal solution (30 μL ONPG 4 mg/mL+150 μL β-Galactosidase buffer(0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 1 mM MgCl₂)) for 20 min. The reactionwas stopped by addition of 50 μL Na₂CO₃ 1 M. Absorbance was read at 405nm.

Luciferase activity was measured using 40 μL cell lysate plus 40 μL ofSteadylite® as described by the manufacturer (Perkin Elmer), on theEnvision (Perkin Elmer).

Omitting OSM was used as a positive control (100% inhibition). Asnegative control 0.5% DMSO (0% inhibition) was used. The positive andnegative controls were used to calculate z′ and ‘percent inhibition’(PIN) values.

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

PIN values were plotted for compounds tested in dose-response and EC₅₀values were derived.

The activity of the compound according to Formula I against JAK-STAT wasdetermined in accordance with the assay described above, thus returningan IC₅₀ value of Non active, >6670, >6670, 8943 (average >7427 nM).

3.2. OSM/IL-1β Signaling Assay

OSM and IL-1β are shown to synergistically upregulate MMP13 levels inthe human chondrosarcoma cell line SW1353. The cells are seeded in 96well plates at 15,000 cells/well in a volume of 120 μL DMEM (Invitrogen)containing 10% (v/v) FBS and 1% penicillin/streptomycin (InVitrogen)incubated at 37° C. 5% CO₂. Cells are preincubated with 15 μL ofcompound in M199 medium with 2% DMSO 1 h before triggering with 15 μLOSM and IL-1β to reach 25 ng/mL OSM and 1 ng/mL IL-1, and MMP13 levelsare measured in conditioned medium 48 h after triggering. MMP13 activityis measured using an antibody capture activity assay. For this purpose,384 well plates (NUNC, 460518, MaxiSorb black) are coated with 35 μL ofa 1.5 μg/mL anti-human MMP13 antibody (R&D Systems, MAB511) solution for24 h at 4° C. After washing the wells 2 times with PBS+0.05% Tween, theremaining binding sites are blocked with 100 μL 5% non-fat dry milk(Santa Cruz, sc-2325, Blotto) in PBS for 24 h at 4° C. Next, the wellsare washed twice with PBS+0.05% Tween and 35 μL of 1/10 dilution ofculture supernatant containing MMP13 in 100-fold diluted blocking bufferis added and incubated for 4 h at room temperature. Next the wells arewashed twice with PBS+0.05% Tween followed by MMP13 activation byaddition of 35 μL of a 1.5 mM 4-Aminophenylmercuric acetate (APMA)(Sigma, A9563) solution and incubation at 37° C. for 1 h. The wells arewashed again with PBS+0.05% Tween and 35 μL MMP13 substrate (Biomol,P-126, OmniMMP fluorogenic substrate) is added. After incubation for 24h at 37° C. fluorescence of the converted substrate is measured in aPerkin Elmer Wallac EnVision 2102 Multilabel Reader (wavelengthexcitation: 320 nm, wavelength emission: 405 nm).

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

3.3. PBL Proliferation Assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 andproliferation is measured using a BrdU incorporation assay. The PBL arefirst stimulated for 72 h with PHA to induce IL-2 receptor, then theyare fasted for 24 h to stop cell proliferation followed by IL-2stimulation for another 72 h (including 24 hr BrdU labeling). Cells arepreincubated with test compounds 1 hr before IL-2 addition. Cells arecultured in RPMI 1640 containing 10% (v/v) FBS.

3.4. Human Whole Blood Assay (hWBA) 3.4.1. Protocol 1 3.4.1.1. IL-6Stimulation Protocol

A flow cytometry analysis was performed to establish JAK1 over JAK2compound selectivity ex vivo using human whole blood. Therefore, bloodis taken from human volunteers who gave informed consent. Blood is thenequilibrated for 30 min at 37° C. under gentle rocking, then aliquotedin Eppendorf tubes. Compound is added at different concentrations andincubated at 37° C. for 30 min under gentle rocking and subsequentlystimulated for 20 min at 37° C. under gentle rocking with interleukin 6(IL-6) for JAK1-dependent pathway stimulation or GM-CSF forJAK2-dependent pathway stimulation. Phospho-STAT1 and phospho-STAT5 arethen evaluated using FACS analysis.

3.4.1.1.1. Phospho-STAT1 Assays 3.4.1.1.1.1. Preparation of Reagents

The 5×Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) was diluted 5-foldwith distilled water and pre-warmed at 37° C. The remaining dilutedLyse/Fix buffer was discarded.

10 μg rhIL-6 (R&D Systems, Cat no 206-IL) was dissolved in 1 mL of PBS0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution wasaliquoted and stored at −80° C.

A 3-fold dilution series of the compound was prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples were incubated with a 1% final DMSO concentration.

3.4.1.1.1.2. Incubation of Blood with Compound and Stimulation with IL-6

Human blood is collected in heparinized tubes. The blood is divided inaliquots of 148.5 μL. Then, 1.5 μL of the test compound dilution isadded to each blood aliquot and the blood samples are incubated for 30min at 37° C. under gentle rocking. One and a half microliter of 10-folddiluted IL-6 stock solution is added to the blood samples (finalconcentration 10 ng/mL) and samples are incubated at 37° C. for 20 minunder gentle rocking.

3.4.1.1.1.3. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer is immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes.

Tubes are centrifuged for 5 min at 400×g at 4° C. The cell pellet iswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletis resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100%methanol is added. Cells are then incubated at 4° C. for 30 min forpermeabilization.

Permeabilized cells are then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μL of 1×PBX containing 3% BSA.

3.4.1.1.1.4. Cell Labeling with Anti Phospho-STAT1 and Anti-CD4Antibodies

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. no 612564 and 559319, respectively) andFITC-conjugated anti-CD4 antibody or control FITC-conjugated isotypeantibody were added and mixed, then incubated for 30 min at 4° C., inthe dark.

Cells were then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

3.4.1.1.1.5. Fluorescence Analysis on FACSCanto II

50,000 total events are counted and Phospho-STAT1 positive cells aremeasured after gating on CD4⁺ cells, in the lymphocyte gate. Data areanalyzed using the FACSDiva software and the percentage of inhibition ofIL-6 stimulation calculated from the percentage of positive cells forphospho-STAT1 on CD4+ cells.

3.4.1.1.2. Phospho-STAT5 Assay 3.4.1.1.2.1. Preparation of Reagents

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) was diluted 5-foldwith distilled water and pre-warmed at 37° C. Remaining diluted Lyse/Fixbuffer was discarded.

10 μg rhGM-CSF (AbCys S.A., Cat no P300-03) was dissolved in 100 μL ofPBS 0.1% BSA to obtain a 100 μg/mL stock solution. The stock solutionwas stored aliquoted at −80° C.

A 3-fold dilution series of the compound was prepared in DMSO (10 mMstock solution). Control-treated samples receive DMSO without the testcompound. All samples were incubated with a 1% final DMSO concentration.

3.4.1.1.2.2. Incubation of Blood with Compound and Stimulation withGM-CSF

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 148.5 μL. Then, 1.5 μL of compound dilution was added toeach aliquot and the blood samples were incubated for 30 min at 37° C.under gentle rocking. A 5,000-fold dilution of the GM-CSF stock solution(1.5 μL) was added to the blood samples (final concentration 20 μg/mL)and samples were incubated at 37° C. for 20 min under gentle rocking.

3.4.1.1.2.3. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer was immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes

Tubes were centrifuged for 5 min at 400×g at 4° C. The cell pellet waswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletwas resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100%methanol was added. Cells were then incubated at 4° C. for 30 min forpermeabilization.

3.4.1.1.2.4. Cell Labeling with Anti Phospho-STAT5 and Anti-CD33Antibodies

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC mouse anti CD33 antibody (BD Biosciences #345800) or control APCmouse IgG1 isotype antibody (BD Biosciences #345818) were added, mixedthen incubated for 30 min at 4° C., in the dark.

Cells were then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

3.4.1.1.2.5. Fluorescence Analysis on FACSCanto II

50,000 total events were counted and Phospho-STAT5 positive cells weremeasured after gating on CD33⁺ cells. Data were analyzed using theFACSDiva software and correspond to the percentage of inhibition ofGM-CSF stimulation calculated from the percentage of positive cells forphosphor-STAT5 on CD33⁺ cells.

3.4.2. Protocol 2 3.4.2.1. Stimulation Protocol

A flow cytometry analysis was performed to establish JAK1 over JAK2compound selectivity ex vivo using human whole blood. Therefore, bloodis taken from human volunteers who gave informed consent. Blood is thenequilibrated for 30 min at 37° C. under gentle rocking, then aliquotedin Eppendorf tubes. Compound is added at different concentrations andincubated at 37° C. for 30 min under gentle rocking and subsequentlystimulated for 20 min at 37° C. under gentle rocking with interleukin 6(IL-6) for JAK1-dependent pathway stimulation, Interferon alpha (IFNα)for JAK1/TYK2 pathway stimulation, interleukin 2 (IL-2) for JAK1/JAK3pathway stimulation or GM-CSF for JAK2-dependent pathway stimulation.Phospho-STAT1 (for IL-6- and IFNα-stimulated cells) and phospho-STAT5(for IL-2- and GM-CSF-stimulated cells) levels are then evaluated usingFACS analysis.

3.4.2.2. Phospho-STAT Assays 3.4.2.2.1. Preparation of Reagents

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. no 558049) was diluted 5-foldwith distilled water and pre-warmed at 37° C. The remaining dilutedLyse/Fix buffer was discarded.

10 μg rhIL-6 (R&D Systems, Cat no 206-IL) was dissolved in 1 mL ofPBS+0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution wasaliquoted and stored at −80° C.

10 μg rhIL-2 (R&D Systems, Cat no 202-IL) was dissolved in 1 mL ofPBS+0.1% BSA to obtain a 10 μg/mL stock solution. The stock solution wasaliquoted and stored at −80° C.

5 μg rhGM-CSF (AbCys S.A., Cat no P300-03) was dissolved in 12.5 mL ofPBS+0.1% BSA to obtain a 400 ng/mL stock solution. The stock solutionwas stored aliquoted at −80° C.

A 3-fold dilution series of the compound was prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples were incubated with a 1% final DMSO concentration.

3.4.2.2. Incubation of Blood with Compound and Stimulation with Triggers

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 148.5 μL. Then, 1.5 μL of the test compound dilution wasadded to each blood aliquot and the blood samples were incubated for 30min at 37° C. under gentle rocking. One and a half microliter of 10-folddiluted IL-6 stock solution, 1.5 μL of uIFNα (PBL Biomedical, Cat no11200-1) stock solution, 1.5 μL of 25-fold diluted IL-2 stock solutionor 1.5 μL of 200-fold dilution of the GM-CSF stock solution was added tothe blood samples and samples were incubated at 37° C. for 20 min undergentle rocking.

3.4.2.2.3. White Blood Cell Preparation

At the end of the stimulation period, 3 mL of 1× pre-warmed Lyse/Fixbuffer was immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes.

Tubes were centrifuged for 5 min at 400×g at 4° C. The cell pellet waswashed with 3 mL of cold 1×PBS, and after centrifugation the cell pelletwas resuspended in 100 μL of ice-cold 1×PBS and 900 μL ice-cold 100%methanol is added. Cells were then incubated at 4° C. for 30 min forpermeabilization.

Permeabilized cells were then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μL of 1×PBX containing 3% BSA.

3.4.2.2.4. Cell Labeling

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. no 612564 and 559319, respectively) andAPC-conjugated anti-CD4 antibody or control APC-conjugated isotypeantibody (BD Biosciences, Cat. no 555349 and 555751, respectively) wereadded to IL-6- and IFNα-stimulated tubes and mixed, then incubated for20 min at 4° C., in the dark.

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC-conjugated anti-CD4 antibody or control APC-conjugated isotypeantibody (BD Biosciences, Cat. no 555349 and 555751, respectively) wereadded to IL-2-stimulated tubes, mixed then incubated for 20 min at 4°C., in the dark.

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG 1K isotype controlantibody (BD Biosciences, Cat. no 612567 and 554680, respectively) andAPC mouse anti CD33 antibody (BD Biosciences #345800) or control APCmouse IgG1 isotype antibody (BD Biosciences Cat. no 345818) were addedto GM-CSF-stimulated tubes, mixed then incubated for 20 min at 4° C., inthe dark.

Cells were then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

3.4.2.2.5. Fluorescence Analysis on FACSCanto II

50,000 total events were counted and Phospho-STAT1 positive cells weremeasured after gating on CD4+ cells, in the lymphocyte gate for IL-6-and IFNα-stimulated cells. Phospho-STAT5 positive cells were measuredafter gating on CD4+ cells, in the lymphocyte gate for IL-2-stimulatedcells. Phospho-STAT5 positive cells were measured after gating on CD33+cells. Data are analyzed using the FACSDiva software and the percentageof inhibition of IL-6 or IFNα stimulation calculated is from thepercentage of positive cells for phospho-STAT1 on CD4+ cells. For theIL-2 stimulated cells, data were analyzed using the FACSDiva softwareand the percentage of inhibition of IL-2 stimulation was calculated fromthe percentage of positive cells for phospho-STAT1 on CD4+ cells. Forthe GM-CSF stimulated cells, the percentage of inhibition of GM-CSFstimulation was calculated from the percentage of positive cells forphosphor-STAT5 on CD33+ cells.

3.4.3. Results

When submitted to these protocols, the compound according to Formula Ireturned a mean IC₅₀ of 11.9 μM on IL-6-induced STAT1 phosphorylation,on 6 different donors. On the IFNα-induced STAT1 phosphorylation, themean IC₅₀ was evaluated to be 15.4 μM on 6 different donors. On theIL-2-induced STAT5 phosphorylation, the mean IC₅₀ was evaluated to be19.6 μM in 5 different donors. On the GM-CSF-induced STAT5phosphorylation, the mean IC₅₀ was evaluated to be over 100 μM in 7different donors.

Example 4 In Vivo Models 4.1. CIA Model 1 4.1.1. Materials

Complete Freund's adjuvant (CFA) and Incomplete Freund's adjuvant (IFA)were purchased from Difco. Bovine collagen type II (CII),lipopolysaccharide (LPS), and Enbrel® was obtained from Chondrex (Isled'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25 mginjectable syringe, France), Whyett (25 mg injectable syringe, France)Acros Organics (Palo Alto, Calif.), respectively. All other reagentsused were of reagent grade and all solvents were of analytical grade.

4.1.2. Animals

Dark Agouti rats (male, 7-8 weeks old) were obtained from HarlanLaboratories (Melderslo, Netherlands). Rats were kept on a 12 hrlight/dark cycle (0700-1900). Temperature was maintained at 22° C., andfood and water were provided ad libitum.

4.1.3. Collagen Induced Arthritis (CIA)

One day before the experiment, CII solution (2 mg/mL) was prepared with0.05 M acetic acid and stored at 4° C. Just before the immunization,equal volumes of adjuvant (IFA) and CII were mixed by a homogenizer in apre-cooled glass bottle in an ice water bath. Extra adjuvant andprolonged homogenization may be required if an emulsion was not formed.0.2 mL of the emulsion was injected intradermally at the base of thetail of each rat on day 1, a second booster intradermal injection (CIIsolution at 2 mg/mL in CFA 0.1 mL saline) was performed on day 9. Thisimmunization method was modified from published methods (Sims et al,2004; Jou et al., 2005).

4.1.4. Study Design

The therapeutic effects of the compounds were tested in the rat CIAmodel. Rats were randomly divided into equal groups and each groupcontained 10 rats. All rats were immunized on day 1 and boosted on day9. Therapeutic dosing lasts from day 16 to day 30. The negative controlgroup was treated with vehicle (MC 0.5%) and the positive control groupwith Enbrel® (10 mg/kg, 3× week., s.c.). A compound of interest wastypically tested at 3 doses, e.g. 6, 10, 60 mg/kg, q.d., p.o.

4.1.5. Clinical Assessment of Arthritis

Arthritis was scored according to the method of Khachigian 2006, Lin etal 2007 and Nishida et al. 2004). The swelling of each of the four pawswas ranked with the arthritic score as follows: 0—no symptoms; 1—mild,but definite redness and swelling of one type of joint such as the ankleor wrist, or apparent redness and swelling limited to individual digits,regardless of the number of affected digits; 2—moderate redness andswelling of two or more types of joints; 3—severe redness and swellingof the entire paw including digits; 4—maximally inflamed limb withinvolvement of multiple joints (maximum cumulative clinical arthritisscore 16 per animal) (Nishida et al., 2004).

To permit the meta-analysis of multiple studies the clinical scorevalues were normalised as follows:

AUC of clinical score (AUC score): The area under the curve (AUC) fromday 1 to day 14 was calculated for each individual rat. The AUC of eachanimal was divided by the average AUC obtained for the vehicle in thestudy from which the data on that animal was obtained and multiplied by100 (i.e. the AUC was expressed as a percentage of the average vehicleAUC per study).

Clinical score increase from day 1 to day 14 (End point score): Theclinical score difference for each animal was divided by the averageclinical score difference obtained for the vehicle in the study fromwhich the data on that animal was obtained and multiplied by 100 (i.e.the difference was expressed as a percentage of the average clinicalscore difference for the vehicle per study).

4.1.6. Change in Body Weight (%) after Onset of Arthritis

Clinically, body weight loss was associated with arthritis (Shelton etal., 2005; Argiles et al., 1998; Rall, 2004; Walsmith et al., 2004).Hence, changes in body weight after onset of arthritis can be used as anon-specific endpoint to evaluate the effect of therapeutics in the ratmodel. The change in body weight (%) after onset of arthritis wascalculated as follows:

${Mice}\text{:}\; \frac{{{Body}\mspace{14mu} {Weight}_{({{week}\mspace{14mu} 6})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\mspace{14mu} 5})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\mspace{14mu} 5})}} \times 100\%$${Rats}\text{:}\; \frac{{{Body}{\mspace{11mu} \;}{Weight}_{({{week}\mspace{14mu} 4})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\mspace{14mu} 3})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\mspace{14mu} 3})}} \times 100\%$

4.1.7. Radiology

X-ray photos were taken of the hind paws of each individual animal. Arandom blind identity number was assigned to each of the photos, and theseverity of bone erosion was ranked by two independent scorers with theradiological Larsen's score system as follows: 0—normal with intact bonyoutlines and normal joint space; 1—slight abnormality with any one ortwo of the exterior metatarsal bones showing slight bone erosion;2—definite early abnormality with any three to five of the exteriormetatarsal bones showing bone erosion; 3—medium destructive abnormalitywith all the exterior metatarsal bones as well as any one or two of theinterior metatarsal bones showing definite bone erosions; 4—severedestructive abnormality with all the metatarsal bones showing definitebone erosion and at least one of the inner metatarsal joints completelyeroded leaving some bony joint outlines partly preserved; 5—mutilatingabnormality without bony outlines. This scoring system was amodification from Salvemini et al., 2001; Bush et al., 2002; Sims etal., 2004; Jou et al., 2005.

4.1.8. Histology

After radiological analysis, the hind paws of mice were fixed in 10%phosphate-buffered formalin (pH 7.4), decalcified with rapid bonedecalcifiant for fine histology (Laboratories Eurobio) and embedded inparaffin. To ensure extensive evaluation of the arthritic joints, atleast four serial sections (5 μm thick) were cut and each series ofsections were 100 μm in between. The sections were stained withhematoxylin and eosin (H&E). Histologic examinations for synovialinflammation and bone and cartilage damage were performed using a doubleblind protocol. In each paw, four parameters were assessed using afour-point scale. The parameters were cell infiltration, pannusseverity, cartilage erosion and bone erosion. Scoring was performedaccordingly, as follows: 1—normal, 2—mild, 3—moderate, 4—marked. Thefour scores were summed together and represented as an additional score,namely the ‘RA total score’.

4.1.9. Micro-Computed Tomography (μCT) Analysis of Calcaneus (Heel Bone)

Bone degradation observed in RA occurs especially at the cortical boneand can be revealed by μCT analysis (Sims N A et al., Arthritis Rheum.50 (2004) 2338-2346: Targeting osteoclasts with zoledronic acid preventsbone destruction in collagen-induced arthritis; Oste L et al., ECTCMontreal 2007: A high throughput method of measuring bone architecturaldisturbance in a murine CIA model by micro-CT morphometry). Afterscanning and 3D volume reconstruction of the calcaneus bone, bonedegradation was measured as the number of discrete objects present perslide, isolated in silico perpendicular to the longitudinal axis of thebone. The more the bone was degraded, the more discrete objects weremeasured. 1000 slices, evenly distributed along the calcaneus (spaced byabout 10.8 μm), were analyzed.

4.1.10. Steady State PK

At day 7 or 11, blood samples were collected at the retro-orbital sinuswith lithium heparin as anti-coagulant at the following time points:predose, 1, 3 and 6 hrs. Whole blood samples were centrifuged and theresulting plasma samples were stored at −20° C. pending analysis. Plasmaconcentrations of each test compound were determined by an LC-MS/MSmethod in which the mass spectrometer was operated in positiveelectrospray mode. Pharmacokinetic parameters were calculated usingWinnonlin® (Pharsight®, United States) and it was assumed that thepredose plasma levels were equal to the 24 h plasma levels.

4.1.11. Results

The compound according to Formula I exhibited statistically significantimprovements in the normalized clinical score values (calculated as AUCor as the difference from day 1 to day 14) at a dose of 60 mg/kg asshown on FIG. 1 and Table I.

TABLE I Rat CIA Clinical Score after treatment with the compoundsdisclosed herein. Day 18 19 20 21 24 25 26 27 28 Vehicle 2.9 3.8 6.0 6.06.4 6.3 6.3 6.3 6.4 Formula II (6 mg/kg) 3.0 3.6 4.2 5.1 5.2 5.1 4.9 5.25.4 Formula I (60 mg/kg) 2.9 4.4 5.0 3.9 4.5 4.3 3.8 3.7 3.3 Formula II3.0 3.6 4.4 4.5 4.5 4.1 3.9 4.3 3.6 (6 mg/kg) + Formula I (60 mg/kg)

4.2. CIA Model 2 4.2.1. Animals

Female Lewis rats (n=76) that weighed 165-194 grams (mean approx. 178 g)on arthritis day 0 were obtained from Charles River Laboratories, Inc.,Wilmington, Mass., (ref#393739). Animals were identified by a distinctnumber at the base of the tail delineating group and animal number.

Upon arrival, animals were housed 4/cage in shoe-box polycarbonate cagesand acclimated for 8 days prior to being immunized with type IIcollagen. No concurrent medications were given.

During the acclimation and study periods, animals were housed in alaboratory environment with temperatures ranging 19-25° F. and relativehumidity of 30%-70%. Automatic timers provided 12 h of light and 12 h ofdark. Animals were allowed access ad libitum to food and water.

4.2.2. Collagen Induced Arthritis (CIA) 4.22.2.1. Preparation

Acclimated female Lewis rats were anesthetized with Isoflurane andreceive subcutaneous/intradermal (SC/ID) injections with 300 μL ofFreund's Incomplete Adjuvant (Difco, Detroit, Mich.) containing 2 mg/mLbovine type II collagen (Elastin Products, Owensville, Mo.) at the baseof the tail and 2 sites on the back on day −9 and on day −3.

Collagen was prepared by making a 4 mg/mL solution in 0.01N Acetic acid.Equal volumes of collagen and Freund's incomplete adjuvant, wereemulsified by hand mixing until a bead of this material holds its formwhen placed in water. Each animal received 300 μL of the mixture on eachoccasion divided over 3 subcutaneous sites on back.

4.2.2.2. Study

On study day 1, onset of arthritis occurred and animals were randomizedinto treatment groups. After randomization, all cages were labelled withprotocol number, group number, and animal numbers. Randomization intoeach group was done after ankle joint swelling was obviously establishedand there was good evidence of bilateral disease.

Animals with established type II collagen arthritis were treated daily(QD) by the oral (PO) route for 13 days with vehicle (methylcellulose0.5% (w/v)), or with active compound(s) prepared in methylcellulose,0.5% (w/v). Oral dosing was initiated on arthritis day 1 and continueddaily (QD at 24 h intervals) through day 13. Animals were terminated onarthritis day 14.

4.2.2.3. Clinical Assessment of Arthritis

The severity of the arthritis was evaluated by measuring the diameter ofboth ankles on the animals. Caliper measurements of ankles were takenevery day beginning on day 0, with a Digitrix II micrometer (Fowler &NSK). Baseline measurements were taken using one ankle with valuesrounded to one-thousandth of an inch. Measurements were confirmed asclinically normal (0.260-0.264 in) by comparison with historical valuesfor rats based on a range of body weights. Baseline measurements werethen applied to both ankles, and these values remain with the animal solong as the ankle was clinically normal with good definition of all theankle bones and no evidence of inflammation. Animals were terminated onday 14.

4.2.2.4. Compounds, Dosage and Results

The compounds were tested individually and in combination at variousdoses, which are listed in Table II below.

TABLE II Compounds and dose tested in the CIA second model CompoundsDose Group control PO, QD — A Vehicle (MC, 0.5%, w/v) 5 mL/kg B FormulaII 1 mg/kg C 3 mg/kg D 10 mg/kg (*) E 30 mg/kg (*) F Formula I 25 mg/kg(*) G 50 mg/kg (*) H Formula II + Formula I 3 mg/kg (Formula II) + I 25mg/kg (Formula I), (*) ((*) = p ≦ 0.05 ANOVA to vehicle control)

4.2.2.5. Conclusion

Ankle diameter measurements indicate that administering the combinationof the compound according to Formula I (25 mg/kg) and Formula II (3mg/kg) provides a stronger effect than the effect obtained with thecompound according to Formula I (25 mg/kg) alone or the compoundaccording to Formula II (3 mg/kg) alone, as shown on FIG. 2 and TableIII below.

TABLE III Evolution of ankle diameter (inches) in the rat CIA modelafter treatment with the compounds disclosed herein. Groups Days A B C DE F G H 0 0.2635 0.2628 0.2625 0.2623 0.2626 0.2620 0.2629 0.2624 10.2635 0.2717 0.2716 0.2718 0.2718 0.2718 0.2717 0.2718 2 0.2635 0.27800.2755 0.2755 0.2744 0.2679 0.2747 0.2705 3 0.2635 0.2865 0.2869 0.28690.2808 0.2683 0.2826 0.2814 4 0.2635 0.2980 0.2990 0.2957 0.2850 0.27080.2892 0.2886 5 0.2635 0.3109 0.3106 0.3058 0.2921 0.2698 0.2973 0.29706 0.2635 0.3246 0.3231 0.3168 0.3000 0.2696 0.3064 0.3061 7 0.26350.3290 0.3296 0.3205 0.3031 0.2706 0.3108 0.3110 8 0.2635 0.3301 0.32910.3259 0.3108 0.2741 0.3150 0.3160 9 0.2635 0.3260 0.3268 0.3216 0.31190.2721 0.3089 0.3085 10 0.2635 0.3227 0.3270 0.3197 0.3122 0.2706 0.30460.3032 11 0.2635 0.3187 0.3185 0.3156 0.3090 0.2711 0.3025 0.3041 120.2635 0.3186 0.3178 0.3165 0.3070 0.2685 0.3006 0.3039 13 0.2635 0.31860.3185 0.3175 0.3069 0.2684 0.2996 0.3014 14 0.2635 0.3216 0.3226 0.31740.3066 0.2679 0.3016 0.2999

Example 5 Pharmacokinetic Studies 5.1. In Vitro Metabolism Study

A study comparing the metabolism of [¹⁴C]-compound according to FormulaII in hepatocytes of mouse, rat, dog and human, clearly demonstratesthat the compound according to Formula II was stable in all species, andconfirms that the extent of metabolism was low in all species, with atleast 80% of the compound according to Formula II after 24 h, as shownon FIG. 3.

Table IV shows the radiometric metabolic profile of the compoundaccording to Formula II, in human and animal species hepatocytes, whichdisplayed one main metabolite (the compound according to Formula I, 11%)and two minor ones (not characterized, <1.0%, visible on chromatograms).

TABLE IV In vitro metabolic profile of the compound according to FormulaII                                     Structure

   

                                    Metabolite2                                    Metabolite3 Mouse 85% 14%  <1.0% <1.0% Rat 86% 12%  <1.0%<1.0% Dog 93%  5.7% <1.0%  1.0% Human 88% 11.2% <1.0% <1.0%

5.2. In Vivo Studies 5.2.1. Single Dose Pharmacokinetics in Animals

Pharmacokinetic studies were performed in various animal species dosedwith the compound according to Formula II to determine the exposure tothe compound according to Formula I as well as its apparent terminalhalf life (T_(1/2)), when possible.

5.2.1.1. Protocol 5.2.1.1.1. Animals

Sprague-Dawley rats (male, 200-250 g), CD1 mice (male, 25-30 g), Beagledogs (male, 9-10 kg), Göttingen minipigs (male, 10-15 kg) and NewZealand White rabbits (male, 3-5 kg) were acclimatized for at least 7days before treatment and were kept on a 12 hr light/dark cycle (07h00-19 h00).

Temperature was maintained at approximately 22° C., and food and waterwere provided ad libitum.

5.2.1.1.2. Pharmacokinetic Study

The compound according to Formula I was formulated in 0.5%methylcellulose and dosed orally to 3 or 6 animals as a singleesophageal gavage at dose ranging from 15 to 180 mg/kg under a dosingvolume of 5 or 10 mL/kg.

The compound according to Formula II was formulated in 0.5%methylcellulose and dosed orally to 3 animals (or 3 mouse/time points)as a single esophageal gavage at dose ranging from 15 to 45 mg/kg undera dosing volume of 5 or 10 mL/kg.

Blood samples were collected over a 24-h period via the jugular vein(dog, minipig), ear blood vessel (rabbit), cardiac puncture (mouse) orretro-orbital sinus (rat) with lithium heparin as anti-coagulant. Wholeblood samples were centrifuged and the resulting plasma samples werestored at −20° C. pending analysis.

5.2.1.1.3. Quantification of Compound Levels in Plasma

Plasma concentrations of the compound according to Formula I and of thecompound according to Formula II were determined by an LC-MS/MS methodwith a limit of quantification ≦10.0 ng/mL for both Formula I andFormula II for all species.

5.2.1.1.4. Determination of Pharmacokinetic Parameters

Pharmacokinetic parameters were calculated using a statistical analysispackage (WinNonLin (Pharsight, Sunnyvale, Calif.: CA 94086, USA).

5.2.1.2. Results 5.2.1.2.1. Result Upon Administration of the CompoundAccording to Formula I Alone

Using the protocol described above, the AUC and apparent terminal halflife were determined as shown below in Table V.

TABLE V Exposure and apparent terminal half life in various species uponadministration of the compound of the invention according to Formula I.Dose AUC Apparent terminal Species (mg/kg) (μg · h/mL) half life (h) Rat20 19.7 (Male) 7 25.2 (Female) 60 102 (Male) 123 (Female) 180 376 (Male)541 (Female) Dog 10 49.4 9 (Male) 25 114 50 290 Rabbit 125 777 NotDetermined (Female) 250 1029

The results presented in Table V show that the compound according toFormula I when dosed on its own is readily absorbed, and thus is exposedin vivo.

5.2.1.2.2. Result Upon Administration of the Compound According toFormula II Alone

Upon administration of the compound according to Formula II, theapparent terminal elimination half-life of the compound according toFormula I was determined in mouse (2.1 h) and rabbit (4.6 h).

5.2.2. Single Dose Pharmacokinetics in Human 5.2.2.1. Protocol

Subjects received the single dose (10, 25, 50, 100, or 200 mg) of thecompound according to Formula II as capsules after a high-fathigh-calorie breakfast (FDA recommendation: a high-fat (approximately 50percent of total caloric content of the meal), and high-calorie(approximately 800 to 1000 calories). An example test meal would be twoeggs fried in butter, two strips of bacon, two slices of toast withbutter, four ounces of hash brown potatoes and eight ounces of wholemilk. Substitutions in this test meal can be made as long as the mealprovides a similar amount of calories from protein, carbohydrate, andfat and has comparable meal volume and viscosity.

Serial blood samples were collected over 72 h to determine plasmaconcentrations of the compounds according to Formula II and Formula Iusing a validated liquid chromatography-mass spectrometry/massspectrometry (LC-MS/MS) method with a limit of quantification of 1.00ng/mL. PK parameters were assessed using a statistical analysis package(WinNonLin (Pharsight, Sunnyvale, Calif.: CA 94086, USA).

5.2.2.2. Plasma Elimination Results

Mean plasma concentration-time profiles of the compound according toFormula II and the compound according to Formula I were measured.

Elimination of the compound according to Formula II over time displayeda biphasic profile with a mean apparent terminal elimination half-lifeof about 5-8 h.

In contrast, the plasma elimination of the compound according to FormulaI displayed a monophasic profile with a mean apparent terminalelimination half-life of ranging from 21 to 27 h.

5.2.2.3. Exposure to the Compound According to Formula II and Formula I

In order to show the comparative exposures of the two compounds, the AUCof the compounds according to Formula I and the compound according toFormula II were calculated. The AUC value represents the total exposureof the compound in the species after dosing. These values were thenexpressed as a ratio of AUC of the compound according to Formula I: AUCof the compound according to Formula II in FIG. 4. From this ratio itcan clearly be seen that there was a significant difference in exposureto a compound according to Formula I after administration of a compoundaccording to Formula II in human compared to the exposure seen inanimals for therapeutically equivalent doses. Without wanting to bebound by theory, it was believed that this difference was related to thedifference in the apparent terminal half-life in the different species.

TABLE VI Ratio of exposure (expressed as AUC) for [Formula I]:[FormulaII] measured on administration of the compound according to Formula II.Species Formula I Formula II Ratio I/II Mouse (30 mg/kg) 11.4 7.3 1.56Rat (45 mg/kg) 13.3 33.3 0.40 Dog (15 mg/kg) 10.6 44.9 0.24 Minipig (30mg/kg) 16.7 19.1 0.88 Rabbit (20 mg/kg) 10 26 0.38 Human (10 mg) 2.890.128 22.58 Human (25 mg) 7.22 0.331 21.81 Human (50 mg) 14.6 0.73519.86 Human (100 mg) 26.9 1.69 15.92 Human (200 mg) 57.6 4.5 12.8

5.2.3. Repeated Dose Pharmacokinetics in Human 5.2.3.1. Protocol

Subjects received repeated dose (25, 50 and 100 mg BID or 200, 300 and450 mg QD) of the compound according to Formula II as capsules after astandard breakfast for 10 days.

Serial blood samples were collected over 12 h (BID regimen) or 24 h (QDregimen) on day 1 and 10 to determine plasma concentrations of thecompounds according to Formula II and Formula I using a validated liquidchromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methodwith a limit of quantification of 1.00 ng/mL for both Formula. PKparameters were assessed using a statistical analysis package (WinNonLin(Pharsight, Sunnyvale, Calif.: CA 94086, USA).

5.2.3.2. Plasma Elimination Results

Mean plasma concentration-time profiles of the compound according toFormula II and the compound according to Formula I were measured.

Elimination of the compound according to Formula II over time displayeda biphasic profile with a mean apparent terminal elimination half-lifeof about 4-11 h.

In contrast, the plasma elimination of the compound according to FormulaI displayed a monophasic profile with a mean apparent terminalelimination half-life of ranging from 22 to 27 h.

5.2.3.3. Exposure to the Compound According to Formula II and Formula I

In order to show the comparative exposures of the two compounds, the AUCof the compounds according to Formula I and the compound according toFormula II were calculated. The AUC value represents the steady stateexposure of the compounds after repeated dosing. The ratio of AUC of thecompound according to Formula I:AUC of the compound according to FormulaII is display in Table VII.

TABLE VII Exposure (expressed as AUC in μg · h/mL) for Formula II andFormula I measured on repeated administration of the compound accordingto Formula II. Regimen BID QD Dose 25 mg 50 mg 100 mg 200 mg 300 mg 450mg Formula II 0.346 0.758 2.38 4.45 4.40 10.2 Formula I 8.66 15.2 41.170.0 66.1 102 Ratio I/II 24.4 20.5 18.3 16.1 15.0 10.1

5.2.4. Conclusion

The compound according to Formula I displays a very similar profile inall species in vitro. However, unexpectedly, in vivo, this profile wasvery different between human and the other animal species, where theapparent terminal half life was at least 3 times higher in human. Thislonger apparent terminal half-life results in accumulation of thecompound according to Formula I in human which offers the possibility ofa wide range of dosage regimen frequency, in particular low frequencydosage regimen, and more particularly from once daily to once every twoweeks, most particularly from once daily to once weekly.

5.3. Target Inhibition

The compound according to Formula II was dosed daily in healthyvolunteers at 200 mg QD and 100 mg BID.

The resulting levels of Formula II and Formula I were then measured andplotted (FIG. 5) as multiples of their respective IC₅₀ as determined bythe Whole Blood assay described above in Example 3.4.

Both Formula II and Formula I were active alone in the CIA rat model.Surprisingly, as shown in FIG. 4, the administration of compound FormulaII is associated with 2 active species, Formula I and Formula II.Without wishing to be bound by theory, it is believed that Formula IIprovides rapid inhibition of the target, and the compound according toFormula I is then steadily formed and accumulated, thus resulting in adrug level above the IC₅₀ level over almost 24 h as shown in thecumulative fold over IC₅₀ on FIG. 6. This was particularly unexpected,as no such profile could have been predicted from the model in rat,mouse and rabbit, where such accumulation does not occur.

TABLE VIII Ratio calculated as [blood circulating dose of the compoundsexpressed as ng/mL]/[circulating dose of the same compounds at IC₅₀quantity]. Formula II at 100 mg BID Formula II at 200 mg QD FormulaFormula Time I + I + point Formula Formula Formula Formula FormulaFormula (h) II I II II I II 0 0.09 0.97 1.06 0.02 0.69 0.71 0.5 0.120.93 1.04 0.46 0.65 1.11 1 0.48 0.88 1.36 3.14 0.69 3.83 2 1.28 0.972.25 3.88 0.84 4.72 3 1.71 1.00 2.71 3.16 0.96 4.12 5 0.88 1.13 2.011.31 1.00 2.31 8 0.26 0.96 1.22 0.36 0.93 1.29 12 0.09 0.86 0.94 — — —12.5 0.12 0.93 1.04 — — — 13 0.48 0.88 1.36 — — — 14 1.28 0.97 2.25 — —— 15 1.71 1.00 2.71 — — — 17 0.88 1.13 2.01 — — — 20 0.26 0.96 1.22 — —— 24 0.09 0.86 0.94 0.11 0.84 0.95

General Conclusions

The data provided in the present application demonstrate that althoughthe compound according to Formula I exhibits similar in vitro potencyagainst JAK1 and JAK2 in a biochemical assay, the in vitro whole bloodselectivity assay, closer to physiological conditions (Saharinen et al.2000 Mol. Cell. Biol., 20(10), 3387), shows that the compound of theinvention according to Formula I exhibits over 10 fold selectivity forJAK1 over JAK2. Furthermore, the compound of the invention according toFormula I shows an unexpected in vivo profile in human compared to whatcould have been anticipated from the in vitro data or from in vivo datain other species. In particular, the compound according to Formula Idisplays a significantly longer apparent terminal half life in humanranging from 21 to 27 h that could not have been predicted by a personof skill in the art, which may result in advantages ranging from lowfrequency dosage regimen and increased patient compliance. Inparticular, the impact of non adherence if the patient misses a dose,might be reduced.

Example 6 Additional Protocols 6.1. Thermodynamic Solubility

A solution of 1 mg/mL of the test compound is prepared in a 0.2Mphosphate buffer pH 7.4 or a 0.1M citrate buffer pH 3.0 at roomtemperature in a glass vial.

The samples are rotated in a Rotator drive STR 4 (Stuart Scientific,Bibby) at speed 3.0 at room temperature for 24 h.

After 24 hrs, 800 μL of the sample is transferred to an eppendorf tubeand centrifuged 5 min at 14000 rpm. 200 μL of the supernatant of thesample is then transferred to a MultiscreenR Solubility Plate(Millipore, MSSLBPC50) and the supernatant is filtered (10-12″ Hg) withthe aid of a vacuum manifold into a clean Greiner polypropylene V-bottom96 well plate (Cat no. 651201). 5 μL of the filtrate is diluted into 95μL (F20) of the same buffer used to incubate in the plate containing thestandard curve (Greiner, Cat no. 651201).

The standard curve for the compound is prepared freshly in DMSO startingfrom a 10 mM DMSO stock solution diluted factor 2 in DMSO (5000 μM) andthen further diluted in DMSO up to 19.5 μM. 3 μL of the dilution seriesas from 5000 μM is then transferred to a 97 μL acetonitrile-buffermixture (50/50). The final concentration range is 2.5 to 150 μM.

The plate is sealed with sealing mats (MA96RD-04S, Kinesis, Cambs, PE198YX, UK) and samples are measured at room temperature on LCMS (ZQ 1525from Waters) under optimized conditions using Quanoptimize to determinethe appropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Ais 15 mM ammonia and solvent B is acetonitrile. The sample is run underpositive ion spray on an XBridge C₁₈ 3.5 μM (2.1×30 mm) column, fromWaters. The solvent gradient has a total run time of 2 min and rangesfrom 5% B to 95% B.

Peak areas are analyzed with the aid of Masslynx software package andpeak areas of the samples are plotted against the standard curve toobtain the solubility of the compound.

Solubility values are reported in μM or μg/mL.

6.2. Aqueous Solubility 6.2.1. Aqueous Solubility 2% DMSO Procedure

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.2M phosphate buffer pH7.4or 0.1M citrate buffer pH 3.0 at room temperature is added.

The final concentration ranges from 200 μM to 2.5 μM in 5 equal dilutionsteps. The final DMSO concentration does not exceed 2%. 200 μM Pyrene isadded to the corner points of each 96 well plate and serves as areference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 hr at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwhich is plotted onto a graph. The first concentration at which thecompound appears completely dissolved is the concentration that isreported below, however the true concentration will lie somewherebetween this concentration and one dilution step higher.

Solubility values measured according to this protocol are reported ing/mL.

6.2.2. Aqueous Solubility 3% DMSO Procedure

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.1M phosphate buffer pH7.4or 0.1M citrate buffer pH3.0 at room temperature is added.

The final concentration will range from 30 μM to 18.75 μM in 5 equaldilution steps. The final DMSO concentration does not exceed 3%. 200 μMPyrene is added to the corner points of each 96 well plate and serves asa reference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 h at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwith a software tool which can be plotted onto a graph. The firstconcentration at which the compound appears completely dissolved is theconcentration reported; however the true concentration lies somewherebetween this concentration and one dilution step higher.

Solubility values measured according to this protocol are reported inμg/mL.

6.3. Plasma Protein Binding (Equilibrium Dialysis)

A 10 mM stock solution of the compound in DMSO is diluted with a factor5 in DMSO. This solution is further diluted in freshly thawed human,rat, mouse or dog plasma (BioReclamation INC) with a final concentrationof 10 μM and final DMSO concentration of 0.5% (5.5 μL in 1094.5 μLplasma in a PP-Masterblock 96well (Greiner, Cat no. 780285))

A Pierce Red Device plate with inserts (ThermoScientific, Cat no. 89809)is prepared and filled with 750 μL PBS in the buffer chamber and 500 μLof the spiked plasma in the plasma chamber. The plate is incubated for 4h at 37° C. while shaking at 230 rpm. After incubation, 120 μL of bothchambers is transferred to 360 μL acetonitrile in a 96-well roundbottom, PP deep-well plates (Nunc, Cat no. 278743) and sealed with analuminum foil lid. The samples are mixed and placed on ice for 30 min.This plate is then centrifuged 30 min at 1200 ref at 4° C. and thesupernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201)for analysis on LCMS.

The plate is sealed with sealing mats (MA96RD-04S) of Kinesis, Cambs,PE19 8YX, UK and samples are measured at room temperature on LCMS (ZQ1525 from Waters) under optimized conditions using Quanoptimize todetermine the appropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Ais 15 mM ammonia and solvent B is acetonitrile. The sample is run underpositive ion spray on an XBridge C₁₈ 3.5 μM (2.1×30 mm) column, fromWaters. The solvent gradient has a total run time of 2 min and rangesfrom 5% B to 95% B.

Peak area from the compound in the buffer chamber and the plasma chamberare considered to be 100% compound. The percentage bound to plasma isderived from these results and is reported as percentage bound toplasma.

The solubility of the compound in the final test concentration in PBS isinspected by microscope to indicate whether precipitation is observed ornot.

6.4. Microsomal Stability 6.4.1. Microsomal Stability 1 h IncubationProcedure

A 10 mM stock solution of compound in DMSO is diluted 1000 fold in a 182mM phosphate buffer pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-incubated at 37° C.

40 μL of deionised water is added to a well of a polypropylene Matrix 2Dbarcode labelled storage tube (Thermo Scientific) and pre-incubated at37° C.

A Glucose-6-phosphate-dehydrogenase (G6PDH) working stock solution isprepared in 182 mM phosphate buffer pH7.4 and placed on ice before use.A co-factor containing MgCl₂, glucose-6-phosphate and NADP+ is preparedin deionised water and placed on ice before use.

A final working solution containing liver microsomes (Xenotech) of aspecies of interest (human, mouse, rat, dog), previously described G6PDHand co-factors is prepared and this mix is incubated for no longer than20 min at room temperature.

30 μL of the pre-heated compound dilution is added to 40μL of pre-heatedwater in the Matrix tubes and 30 μL of the microsomal mix is added.Final reaction concentrations are 3 μM compound, 1 mg microsomes, 0.4U/mL GDPDH, 3.3 mM MgCl₂, 3.3 mM glucose-6-phosphate and 1.3 mM NADP+.

To measure the percentage remaining of compound at time zero, MeOH orMeCN is added (1:1) to the well before adding the microsomal mix. Theplates are sealed with Matrix Sepra seals (Matrix, Cat. No. 4464) andshaken for a few seconds ensure complete mixing of all components.

The samples which are not stopped are incubated at 37° C., 300 rpm andafter 1 hr of incubation the reaction is stopped with MeOH or MeCN(1:1).

After stopping the reaction the samples are mixed and placed on ice for30 min to precipitate the proteins. The plates are then centrifuged 30min at 1200 ref at 4° C. and the supernatant is transferred to a 96v-bottom PP plate (Greiner, 651201) for analysis on LCMS.

These plates are sealed with sealing mats (MA96RD-04S) of Kinesis,Cambs, PE19 8YX, UK and samples are measured at room temperature on LCMS(ZQ 1525 from Waters) under optimized conditions using Quanoptimize todetermine the appropriate mass of the parent molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Ais 15 mM ammonia and solvent B is methanol or acetonitrile, depending onthe stop solution used. The samples are run under positive ion spray onan XBridge C₁₈ 3.5 μM (2.1×30 mm) column, from Waters. The solventgradient has a total run time of 2 min and ranges from 5% B to 95% B.

Peak area from the parent compound at time 0 is considered to be 100%remaining. The percentage remaining after 1 hr incubation is calculatedfrom time 0 and is calculated as the percentage remaining. Thesolubility of the compound in the final test concentration in buffer isinspected by microscope and results are reported.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 60 min.

6.4.2. Microsomal Stability 30 Min Incubation Procedure

A 10 mM stock solution of compound in DMSO is diluted to 6 μM in a 105mM phosphate buffer, pH 7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-warmed at 37° C.

A Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) workingstock solution of 700 U/mL is diluted with a factor 1:700 in a 105 mMphosphate buffer, pH7.4. A co-factor mix containing 0.528M MgCl₂.6H₂O(Sigma, M2670), 0.528M glucose-6-phosphate (Sigma, G-7879) and 0.208MNADP+ (Sigma, N-0505) is diluted with a factor 1:8 in a 105 mM phosphatebuffer, pH7.4.

A working solution is made containing 1 mg/mL liver microsomes(Provider, Xenotech) of the species of interest (human, mouse, rat, dog,. . . ), 0.8 U/mL G6PDH and co-factor mix (6.6 mM MgCl2, 6.6 mMglucose-6-phosphate, 2.6 mM NADP+). This mix is pre-incubated for 15min, but never more than 20 min, at room temperature.

After pre-incubation, compound dilution and the mix containing themicrosomes, are added together in equal amount and incubated for 30 minat 300 rpm. For the time point of 0 min, two volumes of methanol areadded to the compound dilution before the microsome mix is added. Thefinal concentration during incubation are: 3 μM test compound or controlcompound, 0.5 mg/mL microsomes, 0.4 U/mL G6PDH, 3.3 mM MgCl₂, 3.3 mMglucose-6-phosphate and 1.3 mM NaDP+.

After 30 min of incubation, the reaction is stopped with 2 volumes ofmethanol.

Of both time points, samples are mixed, centrifuged and the supernatantis harvested for analysis on LC-MS/MS. The instrument responses (i.e.peak heights) are referenced to the zero time-point samples (as 100%) inorder to determine the percentage of compound remaining. Standardcompounds Propanolol and Verapamil are included in the assay design.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 30 min.

Hepatocyte stability (Concentrations of the compound according toFormula II and its main metabolites (as % of total radioactivity) after24 h incubation of 100 μM [¹⁴C]-Compound according to Formula II inhepatocyte suspension of different species)

6.5. Caco2 Permeability

Bi-directional Caco-2 assays are performed as described below. Caco-2cells are obtained from European Collection of Cell Cultures (ECACC, cat86010202) and used after a 21 day cell culture in 24-well Transwellplates (Fisher TKT-545-020B).

2×105 cells/well are seeded in plating medium consisting ofDMEM+GlutaMAXI+1% NEAA+10% FBS (FetalClone II)+1% Pen/Strep. The mediumis changed every 2-3 days.

Test and reference compounds (propranolol and rhodamine 123 orvinblastine, all purchased from Sigma) are prepared in Hanks' BalancedSalt Solution containing 25 mM HEPES (pH7.4) and added to either theapical (125 μL) or basolateral (600 μL) chambers of the Transwell plateassembly at a concentration of 10 μM with a final DMSO concentration of0.25%.

50 μM Lucifer Yellow (Sigma) is added to the donor buffer in all wellsto assess integrity of the cell layers by monitoring Lucifer Yellowpermeation. As Lucifer Yellow (LY) cannot freely permeate lipophilicbarriers, a high degree of LY transport indicates poor integrity of thecell layer.

After a 1 hr incubation at 37° C. while shaking at an orbital shaker at150 rpm, 70 μL aliquots are taken from both apical (A) and basal (B)chambers and added to 100 μL 50:50 acctonitrilc:water solutioncontaining analytical internal standard (0.5 μM carbamazepine) in a 96well plate.

Lucifer yellow is measured with a Spectramax Gemini XS (Ex 426 nm and Em538 nm) in a clean 96 well plate containing 150 μL of liquid frombasolateral and apical side.

Concentrations of compound in the samples are measured by highperformance liquid-chromatography/mass spectroscopy (LC-MS/MS).

Apparent permeability (Papp) values are calculated from therelationship:

Papp=[compound]acceptor final×Vacceptor/([compound]donorinitial×Vdonor)/Tine×Vdonor/surface area×60×10⁶ cm/s

V=chamber volume

Tine=incubation time.

Surface area=0.33 cm²

The Efflux ratios, as an indication of active efflux from the apicalcell surface, are calculated using the ratio of Papp B>A/Papp A>B.

The following assay acceptance criteria are used:

Propranolol: Papp (A>B) value ≧20(×10⁻⁶ cm/s)

Rhodamine 123 or Vinblastine: Papp (A>B) value <5 (×10⁻⁶ cm/s) withEfflux ratio ≧25.

Lucifer yellow permeability: ≦100 nm/s

6.6. Septic Shock Model

Injection of lipopolysaccharide (LPS) induces a rapid release of solubletumour necrosis factor (TNF-alpha) into the periphery. This model isused to analyse prospective blockers of TNF release in vivo.

Six BALB/cJ female mice (20 g) per group are treated at the intendeddosing once, po. Thirty min later, LPS (15 μg/kg; E. Coli serotype0111:B4) is injected ip. Ninety min later, mice are euthanized and bloodis collected. Circulating TNF alpha levels are determined usingcommercially available ELISA kits. Dexamethasone (5 μg/kg) is used as areference anti-inflammatory compound.

6.7. MAB Model

The MAB model allows a rapid assessment of the modulation of an RA-likeinflammatory response by therapeutics (Khachigian L M. Nature Protocols(2006) 2512-2516: Collagen antibody-induced arthritis). DBA/J mice areinjected i.v. with a cocktail of mAbs directed against collagen II. Oneday later, compound treatment is initiated (vehicle: 10% (v/v) HPβCD).Three days later, mice receive an i.p. LPS injection (50 μg/mouse),resulting in a fast onset of inflammation. Compound treatment iscontinued until 10 days after the mAb injection. Inflammation is read bymeasuring paw swelling and recording the clinical score of each paw. Thecumulative clinical arthritis score of four limbs is presented to showthe severity of inflammation. A scoring system is applied to each limbusing a scale of 0-4, with 4 being the most severe inflammation.

-   -   0 Symptom free    -   1 Mild, but definite redness and swelling of one type of joint        such as the ankle or wrist, or apparent redness and swelling        limited to individual digits, regardless of the number of        affected digits    -   2 Moderate redness and swelling of two or more types of joints    -   3 Severe redness and swelling of the entire paw including digits    -   4 Maximally inflamed limb with involvement of multiple joints

6.8. Oncology Models

In vivo models to validate efficacy of small molecules towardsJAK2-driven myeloproliferative diseases are described by Wernig et al.Cancer Cell 13, 311, 2008 and Geron et al. Cancer Cell 13, 321, 2008.

6.9. Mouse IBD Model

In vitro and in vivo models to validate efficacy of small moleculestowards IBD are described by Wirtz et al. 2007.

6.10. Mouse Asthma Model

In vitro and in vivo models to validate efficacy of small moleculestowards asthma are described by Nials et al., 2008; Ip et al. 2006;Pernis et al., 2002; Kudlacz et al., 2008.

FINAL REMARKS

It will be appreciated by those skilled in the art that the foregoingdescriptions are exemplary and explanatory in nature, and intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, an artisan will recognise apparent modifications andvariations that may be made without departing from the spirit of theinvention. Thus, the invention is intended to be defined not by theabove description, but by the following claims and their equivalents.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

At least some of the chemical names of compounds of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using either ChemDraw® orISIS®/DRAW. Any open valency appearing on a carbon, oxygen or nitrogenatom in the structures herein indicates the presence of a hydrogen atom.Where a chiral center exists in a structure but no specificstereochemistry is shown for the chiral center, both enantiomersassociated with the chiral structure are encompassed by the structure.

REFERENCES

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1. A method for the treatment of a mammal susceptible to or afflictedwith inflammatory bowel disorder, which method comprises administering atherapeutically effective amount of a compound according to Formula I:


2. The method according to claim 1, wherein the said compound isadministered in combination with one or more additional therapeuticagents, wherein said additional therapeutic agent is an inflammatorybowel disorder treating agent.
 3. (canceled)
 4. The method according toclaim 2, wherein the additional therapeutic agent is according toFormula II:


5. The method according to claim 4, wherein the ratio of FormulaI/Formula II is from 1/5 to 1/20.
 6. The method according to claim 4,wherein the ratio of Formula I/Formula II is from 1/5 to 1/10.
 7. Themethod according to claim 1, wherein the mammal is a human. 8.(canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled) 17.(canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)22. (canceled)
 23. (canceled)
 24. The method according to claim 1,wherein the inflammatory bowel disorder is colitis.
 25. The methodaccording to claim 1, wherein the inflammatory bowel disorder is Crohn'sdisease.
 26. The method according to claim 2, wherein the inflammatorybowel disorder is colitis.
 27. The method according to claim 2, whereinthe inflammatory bowel disorder is Crohn's disease.